Triterpenoid sapogenin production in plant and microbial cultures

ABSTRACT

The disclosure relates to a method for enhancing the biosynthesis and/or secretion of sapogenins in the culture medium of plant and microbial cell cultures. Further, the disclosure also relates to the identification of novel genes involved in the biosynthesis of sapogenin intermediates, as well as to novel sapogenin compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. § 371 ofInternational Patent Application PCT/EP2013/059821, filed May 13, 2013,designating the United States of America and published in English asInternational Patent Publication WO 2013/167751 A1 on Nov. 14, 2013,which claims the benefit under Article 8 of the Patent CooperationTreaty and under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication Ser. No. 61/732,817, filed Dec. 3, 2012 and to U.S.Provisional Patent Application Ser. No. 61/645,998, filed May 11, 2012.

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(c) or (e)-SEQUENCE LISTINGSUBMITTED AS PDF FILE WITH A REQUEST TO TRANSFER CRF FROM PARENTAPPLICATION

Pursuant to 37 C.F.R. § 1.821(c) or (e), a file containing a PDF versionof the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.The transmittal documents of this application include a Request toTransfer CRF from the parent application.

TECHNICAL FIELD

The disclosure relates to the fields of plant secondary metabolites withpharmacological or other industrial properties and metabolic engineeringof these phytochemicals. More specifically, the disclosure relates to amethod for enhancing the biosynthesis and/or secretion of sapogeninintermediates in the culture medium of plant and microbial cellcultures. Further, the disclosure also relates to the identification ofnovel genes involved in the biosynthesis of sapogenin intermediates, aswell as to novel sapogenin compounds.

BACKGROUND

Plants synthesize an overwhelming variety of triterpene saponins with anenormous range of biological activities relevant for the pharmaceuticaland chemical industry (e.g., additives to foods and cosmetics). Interestin triterpenoid saponins and its precursors has increased recentlybecause of data showing their diverse biological activities andbeneficial properties, which include antifungal, antibacterial,antiviral, antitumor, molluscicidal, insecticidal, and antifeedantactivities (Suzuki et al. 2002; Sparg et al. 2004; Huhman et al. 2005).Saponins are synthesized by multiple glycosylations of sapogeninbuilding blocks, which, in turn, are produced by multiple modifications(e.g., hydroxylations) of basic sapogenin backbones such as β-amyrin,lupeol, and dammarenediol. These diverse backbones are generated byspecific cyclizations of 2,3-oxidosqualene, which is also anintermediate in the synthesis of membrane sterols. As an illustration,more than seventy saponins have been identified in the model legume, M.truncatula (Huhman and Sumner 2002; Pollier et al. 2011), the core ofthis diversity being centralized in a few aglycones (sapogenins). Also,these precursor sapogenins are very valuable compounds and are importantstarter molecules for further synthetic modifications. For example, thenaturally occurring triterpenoid sapogenin oleanolic acid and itsderivatives possess several promising pharmacological activities, suchas hepato-protective effects, and anti-inflammatory, antioxidant, oranticancer activities (Pollier and Goossens 2012).

The first committed step in triterpenoid saponin biosynthesis is thecyclization of 2,3-oxidosqualene (FIG. 1). This reaction is catalyzed byspecific oxidosqualene cyclases (OSCs), including β-amyrin synthase(bAS; EC 5.4.99.-), and has been functionally characterized in severalplants (Kushiro et al. 1998, Herrera et al. 1998, Iturbe-Ormaetxe et al.2003, Morita et al. 2000, Suzuki et al. 2002). Then, the action ofoxidative enzymes (typically cytochrome P450 monooxygenases or CYPs) andglycosyltransferases convert β-amyrin to various triterpene saponins indifferent plant species. For example, subsequent modifications thatimpart functional properties and diversify the basic triterpenoidbackbone include the addition of small functional groups, includinghydroxyl, keto, aldehyde, and carboxyl moieties, generally followed byglycosylation reactions (Augustin et al. 2011). To date, a number ofCYPs that use β-amyrin as a substrate have been identified indicotyledonous plants, whereas just one (CYP51H10) has been identifiedin monocots.

Present availability of saponins and sapogenins depends on theirextractability from plants and is often uneconomical and inefficient.Often, laborious extraction schemes have to be developed for eachspecific metabolite of interest and a steady supply of sufficientamounts of specific sapo(ge)nins from plants that accumulate mixtures ofstructurally related compounds is not feasible. Synthetic chemistrymainly attempts to address these issues by chemically linking desiredside chains to extracted sapogenins, as was done for oleanolic acid.However, the structural complexity of the sapogenins hampers chemicalsynthesis and the availability of corresponding sapogenins forms a majorbottleneck.

The culture of plant cells has been explored since the 1960s as a viablealternative for the production of complex phytochemicals of industrialinterest. For example, the use of large-scale plant cell cultures inbioreactors for the production of alkaloids has been extensively studied(Verpoorte et al. 1999). Despite the promising features anddevelopments, the production of plant-derived pharmaceuticals by plantcell cultures has not been fully commercially exploited. The mainreasons for this reluctance shown by industry to produce phytochemicalsby means of cell cultures, compared to the conventional extraction ofwhole plant material, are economical ones based on the slow growth andthe low production levels of phytochemicals by such plant cell cultures.Important causes are the toxicity of such compounds to the plant cell,and the role of catabolism of these compounds. Another important problemis that many phytochemicals, such as the triterpene saponins and itsprecursors, are mostly retained intracellularly, complicating thedownstream processing and purification. Another important problem isthat for many phytochemicals, the precursors or intermediates in thepathway do not accumulate or only in trace amounts, because they arereadily converted by the downstream enzymes.

Biotechnological production of either complete saponins, or of sapogeninpathway intermediates that are not readily accessible, may circumventthe limitation of natural sapo(ge)nin availability. However,circumvention of laborious and uneconomical extraction procedures forindustrial production from plants is also hampered by lack of knowledgeand availability of genes in saponin biosynthesis. As a consequence,although triterpene synthases have been expressed in microbial hostssuch as Saccharomyces cerevisiae, there has been little effort made sofar to engineer the metabolism of a microbial host for enhancedproduction of triterpenes. By contrast, there have been manyconsiderable efforts to engineer microbes for higher production ofmono-, sesqui- and diterpenes. Notably, triterpene production may not beas amenable to engineering efforts as the volatile sesquiterpenes andmonoterpenes that readily diffuse out of the cell.

Therefore, a need exists for the cost-effective biotechnologicalproduction of high value sapo(ge)nins or other triterpene buildingblocks in a convenient host cell.

BRIEF SUMMARY

Evidence is available that sapogenins, when produced in their naturalhosts (plants), are often only found in trace amounts intracellularly inplant cells, as also demonstrated in Example 1. Moreover, and as shownin Examples 3 and 4, although sapogenins can be heterologously producedin genetically engineered yeast cells, they are only detected whenextracted from the cells, and they are not found in the growth medium.In order to overcome these problems, it has been found that byincubating a eukaryotic cell culture that is capable of intracellularlysynthesizing sapogenins in a culture medium with cyclodextrins,significant amounts of sapogenins can be extracted from the culturemedium (Examples 5, 6 and 7). Cyclodextrins, which are cyclicoligosaccharides consisting of five or more α-D-glucopyranose residueslinked by a(1→4) glucosidic bonds, are known to act as sequesteringagents of phytosterols from membranes (Raffaele et al. 2009) and havebeen used as elicitors to increase the production and extraction ofphytosterols from cultures of plant cells (EP2351846; Sabater-Jara etal. 2010). In the disclosure, it is shown that intracellularlysynthesized sapogenins can be released in the growth medium ofeukaryotic cell cultures in the presence of cyclodextrins and canaccumulate in significant amounts. In addition, it was shown thatsubstantially higher amounts can be obtained by repeatedly addingcyclodextrins to the culture medium.

Thus, in a first aspect, the disclosure relates to a method forproducing triterpenoid sapogenins in the extracellular medium of aeukaryotic cell culture comprising:

-   -   a. providing eukaryotic cells capable of synthesizing        triterpenoid sapogenins under suitable conditions;    -   b. incubating the cells in culture medium comprising        cyclodextrins; and    -   c. optionally, extracting the sapogenins from the culture        medium.

In certain embodiments, the eukaryotic cells naturally producetriterpenoid sapogenins, such as plants cells. Alternatively, theeukaryotic cells are genetically engineered to produce triterpenoidsapogenins. Such genetically engineered eukaryotic cells can bemicrobial cells, such as yeast cells, or plant cells, or algal cells.

In certain embodiments, the cyclodextrins are selected from the groupcomprising randomly methylated cyclodextrins or hydroxypropylatedcyclodextrins. Preferably, the cyclodextrin is a β-cyclodextrin.According to particular embodiments, the cyclodextrins may be added tothe culture medium once or at different consecutive time points.

In other aspects, the disclosure also envisages eukaryotic cellsgenetically engineered to synthesize sapogenins.

Further, a sapogenin obtained by any of the above-described methods isalso encompassed.

According to yet another aspect, the disclosure relates to an isolatedpolypeptide selected from the group consisting of:

-   -   (a) a polypeptide encoded by a polynucleotide comprising SEQ ID        NO:1 or 2;    -   (b) a polypeptide comprising a polypeptide sequence having a        least 75% identity to the polypeptide encoded by a        polynucleotide sequence having SEQ ID NO:1 or 2;    -   (c) a polypeptide comprising an amino acid sequence as set forth        in SEQ ID NO:3 or 4;    -   (d) a polypeptide comprising an amino acid sequence with at        least 75% identity to SEQ ID NO:3 or 4;    -   (e) fragments and/or variants of the polypeptides according to        (a), (b), (c), or (d).

In one embodiment, the disclosure relates to any of the above-describedpolypeptides wherein the polypeptide sequence is consisting of an aminoacid sequence as set forth in SEQ ID NO:3 or 4 and polypeptide sequenceshaving at least 75% identity to SEQ ID NO:3 or 4.

According to yet another aspect, the disclosure relates to an isolatedpolynucleotide selected from the group consisting of:

-   -   (a) a polynucleotide comprising a polynucleotide sequence having        the sequence SEQ ID NO:1 or 2;    -   (b) a polynucleotide comprising a polynucleotide sequence having        at least 70% identity to the sequence having SEQ ID NO:1 or 2;    -   (c) a polynucleotide that encodes the polypeptide sequence as        set forth in SEQ ID NO:3 or 4;    -   (d) a polynucleotide that encodes the polypeptide sequence as        set forth in SEQ ID NO:3 or 4;    -   (e) fragments and variants of the polynucleotides according to        (a), (b), (c) or (d).

In a specific embodiment, the disclosure relates to a chimeric genecomprising the following operably linked sequences: a) a promoter regioncapable of directing expression in a eukaryotic cell (as definedhereinbefore); b) a DNA region encoding a polypeptide as defined above;and c) a 3′ polyadenylation and transcript termination region.

A vector comprising a polynucleotide sequence or a chimeric gene asdefined above also forms part of the disclosure, as well as a host cellcomprising a polynucleotide sequence or a chimeric gene or a vector asdefined above.

According to a particular aspect, the disclosure provides a transgenicplant or a cell derived thereof that is transformed with theabove-described vector.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1: Plant bioactive triterpene saponins. Schematic summary ofbiosynthetic pathways and variability in structure. Single and multiplearrows indicate single and multiple catalytic (enzymatic orsemi-synthetic) conversions, respectively. AT, acyltransferase; BAS,(3-amyrin synthase; CAS, cycloartenol synthase; CytP450, CytochromeP450; FPP, farnesylpyrophosphate; GT, glycosyltransferase; LUP, lupeolsynthase; MT, methyltransferase; PNA, dammarenediol-II synthase; SQE,squalene epoxidase; and SQS, squalene synthase.

FIG. 2: GC chromatogram of extraction from M. truncatula hairy rootsshowing the presence of abundant sterols and sterol intermediates, butonly trace amounts of triterpenoid sapogenins.

FIG. 3: GC chromatogram of: Panel A) extraction from cells of strain TM3showing β-amyrin at 27.2 min, Panel B) extraction from spent medium ofstrain TM3, Panel C) extraction from cells of strain TM5, and Panel D)β-amyrin standard.

FIG. 4: GC chromatogram of: Panel A) extraction from cells of strain TM6showing lupeol at 28.9 min, Panel B) extraction from spent medium ofstrain TM6, Panel C) extraction from cells of strain TM5, and Panel D)lupeol standard.

FIG. 5: Quantification of: Panel A) β-amyrin from the cells and spentmedium of strain TM3, where 100% corresponds to 36.2 mg/L of β-amyrin,and Panel B) lupeol from the cells and spent medium of strain TM6, where100% corresponds to 46.3 mg/L of lupeol. Higher concentrations ofβ-amyrin and lupeol were quantified from the extracts of spent mediumcompared to cell pellet upon cyclodextrin treatment.

FIG. 6: Dose-dependent secretion of β-amyrin quantified for strain TM3,where cyclodextrin was added on: Day 1 (I1), Day 1 and 2 (I12), Day 1, 2and 3 (I3), Day 2 (R1), Day 2 and 3 (R2), Day 3 (AR) and untreatedcontrol (C).

FIG. 7: GC chromatograms of extraction performed on spent medium of:Panel A) strain TM10 expressing CYP716A12, Panel B) strain TM11expressing CYP88D6, Panel C) strain TM12 expressing CYP93E2, and PanelD) control strain TM26 expressing no CYP.

FIG. 8: GC chromatograms of extraction performed on spent medium of:Panel A) strain TM22 expressing CYP716A12, and Panel B) control strainTM28 expressing no CYP.

FIG. 9: GC chromatograms of extraction performed on spent medium of M.truncatula hairy roots treated for 48 hours with: Panel A) 25 mMcyclodextrin, Panel B) 25 mM cyclodextrin and 100 μM methyl jasmonate,Panel C) 100 μM methyl jasmonate, and Panel D) untreated control.

FIG. 10: Chemical structure of oleanane-type sapogenin backbone.Asterisks (*) indicate the carbon positions for which a CytP450 hasalready been characterized.

FIG. 11: Transcript profiling of jasmonate elicited B. falcatum roots.Subcluster of the B. falcatum transcriptome, comprising tagscorresponding to genes reported to be involved in triterpenebiosynthesis, or with high sequence similarity to such genes. Treatmentsand time points (in hours) are indicated on top. Blue and yellow boxesreflect transcriptional activation and repression relative to theaverage expression level, respectively. Gray boxes correspond to missingtime points. The arrowhead indicates the CytP450 functionally defined inthis study.

FIG. 12: GC chromatograms corresponding to: Panel A) Extraction fromcells of strain TM7. Panel B) Extraction from cells of strain TM10.Enclosed box figure shows the mass spectra extracted from the indicated(*) peak. Panel C) Extraction from cells of control strain TM26. PanelD) Mass spectra extracted from the peak indicated (*) at 31.8 minutes ofstrain TM7. Panel E) Mass spectra of an erythrodiol standard.

FIG. 13: Panel A) Effect of CPR:CytP450 ratio on the in vivo activity ofCYP716AO21 in strains TM8, TM9 and TM7. Panel B) Relative amounts ofhydroxylated β-amyrin quantified from the cells and spent medium ofstrain TM9. Panel C) Relative amounts of hydroxylated β-amyrinquantified from spent medium of strain TM9 treated with differentvariants of CD.

FIG. 14: Chemical structure of maesasapogenins.

FIG. 15: Transcript profiling of jasmonate elicited M. lanceolataplants. Subcluster of the M. lanceolata transcriptome, comprising alltags corresponding to genes reported to be involved in terpenebiosynthesis, or with high sequence similarity to such genes, and allgene tags corresponding to CytP450s. Treatments and time points (inhours) are indicated on top. Blue and yellow boxes reflecttranscriptional activation and repression relative to the averageexpression level, respectively. Gray boxes correspond to missing timepoints.

FIG. 16: GC chromatograms corresponding to: Panel A) Extraction fromspent medium of strain TM21. Arrowheads indicate the positions thatcould be hydroxylated by ML593 and are common with predicted positionsof CYP716AO21. Panel B) Extraction from spent medium of strain TM9.Panel C) Extraction from spent medium of control strain TM27. Rightpanel shows mass spectra extracted from the indicated (*) peaks.

FIG. 17: GC chromatograms corresponding to: Panel A) Extraction fromspent medium of strain TM30. Panel B) Extraction from spent medium ofstrain TM9. Panel C) Extraction from spent medium of strain TM17. PanelD) An echinocystic acid standard. Panel E) Mass spectra extracted fromthe peak indicated (*) at 40.5 minutes of strain TM30. Parts of thestructure in blue depict the possible unknown hydroxylation position.Panel F) Mass spectra extracted from echinocystic acid standard. PanelG) Oxidation of β-amyrin by CYP716AO21 and CYP716A12.

FIG. 18: GC chromatograms corresponding to: Panel A) Extraction fromspent medium of strain TM31. Panel B) Extraction from spent medium ofstrain TM17. Panel C) Extraction from spent medium of strain TM21. PanelD) Extraction from spent medium of strain TM30. Panel E) An echinocysticacid standard. Right panel shows the mass spectra of indicated (*)peaks. Parts of structure highlighted in blue indicate probablehydroxylation positions.

FIG. 19: GC chromatograms corresponding to: Panel A) Extraction fromspent medium of strain TM32. Panel B) Extraction from spent medium ofstrain TM21. Panel C) Extraction from spent medium of strain TM18. PanelD) Extraction from spent medium of control strain TM27. Panel E) Massspectra extracted from indicated (*) peaks of strain TM18 and TM32.Right panels show the mass spectra extracted from the indicated (*)peaks. Panel F) Oxidation of β-amyrin by ML593 and CYP88D6.

FIG. 20: GC chromatograms corresponding to: Panel A) Extraction fromspent medium of strain TM33. Panel B) Extraction from spent medium ofcontrol strain TM5. Panel C) A β-amyrin standard. Panel D) A α-amyrinstandard. Right panels show the mass spectra extracted from theindicated (*) peaks. Panel E) Cyclization of 2,3-oxidosqualene byα-amyrin synthase (aAS), β-amyrin synthase (bAS), and dammarenediolsynthase (DDS) to α-amyrin, β-amyrin and dammarenediol, respectively.

FIG. 21: GC chromatograms corresponding to: Panel A) Extraction fromspent medium of strain TM37. Right panels show the mass spectraextracted from the indicated (*) peaks. Panel B) Extraction from spentmedium of control strain TM38. Parts of structure in green indicate theputative hydroxylation position.

DETAILED DESCRIPTION

The disclosure will be described with respect to particular embodimentsand with reference to certain drawings; the disclosure is not limitedthereto but only by the claims. Any reference signs in the claims shallnot be construed as limiting the scope. The drawings described are onlyschematic and are non-limiting. In the drawings, the size of some of theelements may be exaggerated and not drawn on scale for illustrativepurposes. Where the term “comprising” is used in this description andclaims, it does not exclude other elements or steps. Where an indefiniteor definite article is used when referring to a singular noun, e.g., “a”or “an,” “the,” this includes a plural of that noun unless somethingelse is specifically stated. Furthermore, the terms “first,” “second,”“third,” and the like, in the description and in the claims, are usedfor distinguishing between similar elements and not necessarily fordescribing a sequential or chronological order. It is to be understoodthat the terms so used are interchangeable under appropriatecircumstances and that the embodiments of the disclosure describedherein are capable of operation in sequences other than described orillustrated herein.

Unless otherwise defined herein, scientific and technical terms andphrases used in connection with the disclosure shall have the meaningsthat are commonly understood by those of ordinary skill in the art.Generally, nomenclatures used in connection with, and techniques ofmolecular and cellular biology, genetics and protein and nucleic acidchemistry and hybridization described herein are those well-known andcommonly used in the art. The methods and techniques of the disclosureare generally performed according to conventional methods well known inthe art and as described in various general and more specific referencesthat are cited and discussed throughout this specification unlessotherwise indicated. See, for example, Sambrook et al., MolecularCloning: A Laboratory Manual, 2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989); Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates (1992, andSupplements to 2002); and Leach, Molecular Modelling: Principles andApplications, 2d ed., Prentice Hall, New Jersey (2001).

According to a first aspect, the disclosure relates to a method forproducing triterpenoid sapogenins in the extracellular medium of aeukaryotic cell culture comprising:

-   -   a. providing eukaryotic cells capable of synthesizing        triterpenoid sapogenins under suitable conditions; and    -   b. incubating the cells in culture medium comprising        cyclodextrins; and    -   c. optionally, extracting the sapogenins from the culture        medium.

Saponins are a group of natural bio-active compounds that consist of anisoprenoidal-derived aglycone, designated “genin” or “sapogenin,”covalently linked to one or more sugar moieties. This combination ofpolar and non-polar structural elements in their molecules explainstheir soap-like behavior in aqueous solutions. Most known saponins areplant-derived secondary metabolites, though several saponins are alsofound in marine animals such as sea cucumbers and starfish. In plants,saponins are generally considered to be part of defense systems due toanti-microbial, fungicidal, allelopathic, insecticidal andmolluscicidal, etc., activities. Typically, saponins reside inside thevacuoles of plant cells. Extensive reviews on molecular activities,biosynthesis, evolution, classification, and occurrence of saponins aregiven by, e.g., Augustin et al. (2011) and Vincken et al. (2007). Thus,the term “sapogenin,” as used herein, refers to an aglycone, ornon-saccharide, moiety of the family of natural products known assaponins.

The commonly used nomenclature for saponins distinguishes betweentriterpenoid saponins (also: triterpene saponins) and steroidalsaponins, which is based on the structure and biochemical background oftheir aglycones. Both sapogenin types are thought to derive from2,3-oxidosqualene, a central metabolite in sterol biosynthesis. Inphytosterol anabolism, 2,3-oxidosqualene is mainly cyclized intocycloartenol. “Triterpenoid sapogenins” branch off the phytosterolpathway by alternative cyclization of 2,3-oxidosqualene, while“steroidal sapogenins” are thought to derive from intermediates in thephytosterol pathway downstream of cycloartenol formation (see also FIG.1). A more detailed classification of saponins based on sapogeninstructure with 11 main classes and 16 subclasses has been proposed byVincken et al. (2007; particularly from page 276 to page 283, and alsoFIGS. 1 and 2), which is all incorporated herein by reference. Inparticular, saponins may be selected from the group comprisingdammarane-type saponins, tirucallane-type saponins, lupane-typesaponins, oleanane-type saponins, taraxasterane-type saponins,ursane-type saponins, hopane-type saponins, cucurbitane-type saponins,cycloartane-type saponins, lanostane-type saponins, and steroid-typesaponins. The aglycon backbones, the sapogenins, can be similarlyclassified and may be selected from the group comprising dammarane-typesapogenins, tirucallane-type sapogenins, lupane-type sapogenins,oleanane-type sapogenins, taraxasterane-type sapogenins, ursane-typesapogenins, hopane-type sapogenins, cucurbitane-type sapogenins,cycloartane-type sapogenins, lanostane-type sapogenins, and steroid-typesapogenins. Examples of sapogenins produced in plants are given in Table1.

According to the above definitions, and as used herein, the“triterpenoid sapogenins” may be selected from the group comprisingdammarane-type sapogenins, tirucallane-type sapogenins, lupane-typesapogenins, oleanane-type sapogenins, ursane-type sapogenins, andhopane-type sapogenins. Thus, according to specific embodiments, thetriterpenoid sapogenins as produced by the method of the disclosure aredammarane-type sapogenins, or tirucallane-type sapogenins, orlupane-type sapogenins, or oleanane-type sapogenins, or ursane-typesapogenins, or hopane-type sapogenins.

Triterpenoid sapogenins typically have a tetracyclic or pentacyclicskeleton. As described in the Background section, the sapogenin buildingblocks themselves may have multiple modifications, e.g., smallfunctional groups, including hydroxyl, keto, aldehyde, and carboxylmoieties, of precursor sapogenin backbones such as β-amyrin, lupeol, anddammarenediol.

The terms “triterpene” and “triterpenoid” are used interchangeablyherein.

It is to be understood that the triterpenoid sapogenins, as used herein,also encompass new-to-nature triterpenoid compounds, which arestructurally related to the naturally occurring triterpenoid sapogenins.These new-to-nature triterpenoid sapogenins may be currentlyunextractable compounds by making use of existing extraction proceduresor may be novel compounds that can be obtained after genetic engineeringof the synthesizing eukaryotic host cell (see further herein).

For the sake of clarity, the term “triterpenoid sapogenins,” as usedherein, is not meant to cover phytosterols or phytosterol pathwayintermediates. Phytosterols, which encompass plant sterols and stanols,are triterpenes that are important structural components of plantmembranes and free phytosterols serve to stabilize phospholipid bilayersin plant cell membranes just as cholesterol does in animal cellmembranes. Stanols are a fully saturated subgroup of phytosterols(contain no double bonds). Non-limiting examples of phytosterols, whichform important structural components of plant membranes, arestigmasterol, β-sitosterol, fucosterol, and campesterol.

TABLE 1 Triterpenoid sapogenins comprising the core of commonlyaccumulating saponins produced by plants. Sapogenins comprised incommonly Chemical formula Plant genus accumulating saponins of sapogeninReference Medicago 2,3-dihydro-23-oxoolean-12-en-28-oic C₃₀H₄₆O₅ (Tavaet al., acid 2011) Medicagenic acid C₃₀H₄₆O₆ Zanhic acid C₃₀H₄₆O₇Oleanolic acid; Soyasapogenol E C₃₀H₄₈O₃ Hederagenin, 2-hydroxyoleanolicacid; C₃₀H₄₈O₄ Queretaroic acid Bayogenin; 2-hydroxyqueretaroic acid;C₃₀H₄₈O₅ Caulophyllogenin Sophoradiol; 3,24-dihydroolean-12-ene C₃₀H₅₀O₂Soyasapogenol B C₃₀H₅₀O₃ Soyasapogenol A C₃₀H₅₀O₄ Panax DammarenediolC₃₀H₅₂O₂ (Zou et al., Panaxadiol; Protopanaxadiol C₃₀H₅₂O₃ 2002)Panaxatriol; Protopanaxatriol C₃₀H₅₂O₄ Bupleurum Rotundioside Osapogenin C₃₀H₄₆O₄ (Ashour and Rotundioside L, M sapogenin C₃₀H₄₆O₅Wink, 2011) Sandrosapogenin III, VIII C₃₀H₄₆O₆ Sandrosapogenin IVC₃₀H₄₆O₇ Saikogenin C, M; Rotundioside B, C C₃₀H₄₈O₃ sapogenin;Sandrosapogenin IX, X; Rotundifolioside A, I, J sapogenin Saikogenin A,B1, B2, D, G, K, N, O, P, C₃₀H₄₈O₄ S; Prosaikogenin A, H; RotundiosideA, J, K, Q, S, V sapogenin; Rotundifolioside D, E, F, G, HSandrosapogenin II, V, VI; C₃₀H₄₈O₅ Bupleuroside VI sapogenin;Saikosapogenin L, Q, Q2, R, U, V2, V; Scorzoneroside A, B, C sapogeninRotundioside D sapogenin C₃₀H₅₀O₃ Rotundifolioside B, C C₃₀H₅₀O₄Hydroxysaikosapogenin A, C, D; C₃₀H₅₀O₅ Bupleuroside VIII sapogeninRotundioside N sapogenin C₃₀H₅₀O₅ Saikosapogenin F C₃₀H₅₂O₃Methoxysaikosapogenin F; C₃₀H₅₂O₄ Saikosapogenin T; Bupleuroside IXsapogenin; Rotundioside X, Y sapogenin Saikogenin B3, B4; RotundiosideP, R, C₃₀H₅₂O₅ U sapogenin Maesa 16-oxo-28-hydroxyolean-12-ene C₃₀H₄₈O₃(Manguro et 16,22-dihydroxyolean-12-en-28-al C₃₀H₄₈O₄ al., 2011)16,21,22-trihydroxyoleanane-13:28- C₃₀H₄₈O₆ ollide16,28-dihydroxyolean-12-ene C₃₀H₅₀O₃ 16,22,28-trihydroxyolean-12-eneC₃₀H₅₀O₄ 16,21,22,28-tetrahydroxyolean-12-ene C₃₀H₅₀O₅ Maesasapogenin I,II, III, IV, V, VI, VII C₃₀H₅₀O₆ Saponaria Quillaic acid C₃₀H₄₆O₅ (Guoet al., 1998) Betula Betulin C₃₀H₄₈O₂ (Rickling and Betulinic acidC₃₀H₄₈O₃ Glombitza, Betulafolientetraol C₃₀H₅₂O₅ 1993) OleanderOleanolic acid; Ursolic acid C₃₀H₄₈O₃ (Stiti et al, Maslinic acidC₃₀H₄₈O₄ 2007) Bacchara-12,21-dien-3-ol C₃₀H₄₈O Butyrospermol C₃₀H₅₀O

The method of the disclosure makes use of cyclodextrins for theproduction of triterpenoid sapogenins in the culture medium ofeukaryotic cells that are capable of synthesizing triterpenoidsapogenins. With “cyclodextrins” (CDs) (sometimes also calledcycloamyloses) are meant cyclic oligosaccharides composed of five ormore (α-1,4)-linked α-D-glucopyranose subunits, which are well-known inthe art. As used herein, “cyclodextrins” encompass both naturallyoccurring cyclodextrins as well as chemical derivatives thereof, asdescribed further herein. Cyclodextrins possess a cage-likesupramolecular structure, and are capable of forming inclusion complexeswith a variety of guest molecules: CDs incorporate compounds in theirhydrophobic cavities depending on the cavity size. The most typicalcyclodextrins contain a set of six to eight glucopyranoside units in aring (the cyclodextrin core), creating a cone shape. Within this family,α-cyclodextrins (αCD) have six glucopyranoside units, β-cyclodextrins(βCD) have seven glucopyranoside units, and γ-cyclodextrins (γCD) haveeight glucopyranoside units in a ring. Each glucopyranoside unit has,according to the standard atom numbering system, one primary alcoholgroup at carbon 6 and two secondary alcohol groups at carbons 2 and 3.These natural cyclodextrins, in particular βCD, are of limited aqueoussolubility. Therefore, several derivatives of cyclodextrins have beendeveloped. Numerous chemical modifications of cyclodextrins are known inthe art, as summarized, for instance, by A. Croft and R. Bartsch inTetrahedron Report No. 147, Tetrahedron (1983) 39(9):1417-1474, andwhich is incorporated herein by reference. These derivatives usually areproduced by aminations, esterifications or etherifications of primaryand secondary hydroxyl groups of the cyclodextrins. Depending on thesubstituent, the solubility of the cyclodextrin derivatives is usuallydifferent from that of their parent cyclodextrins. Virtually allderivatives have a changed hydrophobic cavity volume and also thesemodifications can improve solubility, stability against light or oxygenand help control the chemical activity of guest molecules. For example,and without the purpose of being limitative, water-soluble cyclodextrinderivatives of commercial interest include the hydroxypropyl derivativesof βCD and γCD, the randomly methylated β-cyclodextrin (RMβCD), andsulfobutylether β-cyclodextrin sodium salt (SBEβCD).

Thus, according to a preferred embodiment, the cyclodextrin that is usedin the method of the disclosure is chosen from the group comprisingrandomly methylated cyclodextrin or hydroxypropylated cyclodextrin.Preferably, the degree of substitution by methyls per glucose unit ofthe randomly methylated cyclodextrin is between one and three, and morepreferably, the degree of substitution by methyls per glucose unit ofthe randomly methylated cyclodextrin is two. Preferably, the degree ofsubstitution by hydroxypropyls per glucose unit of the hydroxypropylatedcyclodextrin is between 0.6 and 0.9. According to another preferredembodiment, the cyclodextrin that is used in the method of thedisclosure is a β-cyclodextrin. According to still another embodiment,the cyclodextrin that is used in the method of the disclosure is amethylated β-cyclodextrin.

In another embodiment, the concentration of cyclodextrins in the culturemedium is less than 25 mM, preferably less than 10 mM, more preferablybetween 2 and 7 mM. According to more specific embodiments, theconcentration of cyclodextrins in the culture medium is 5 mM or 2.5 mM,or 1 mM. In one other embodiment, cyclodextrins are added to the culturemedium at one point in time, for example, immediately before or afterinoculation with eukaryotic cells. Preferably, cyclodextrins are addedto the culture medium at different consecutive time points, for example,immediately before or after inoculation with eukaryotic cells, and thenon a daily basis after the first addition of cyclodextrins, or everyother day after the first addition of cyclodextrins. This is furtherillustrated, without the purpose of being limitative, in Example 5.

With “production” of triterpenoid sapogenins is meant both intracellularproduction as well as secretion into the medium. According to apreferred embodiment, the triterpenoid sapogenins as produced by themethod of the disclosure are secreted into the extracellular medium. Theproduction of triterpenoid sapogenins typically is enhanced or inducedby using the method of the disclosure. An “enhanced production” of atriterpenoid sapogenin means that there exists already a detectableamount of this metabolite in the eukaryotic cell in the absence ofcyclodextrins but that detection only becomes possible in theextracellular medium upon adding cyclodextrins according to thedisclosure. An “induced production” of a triterpenoid sapogenin meansthat there is no detectable production of this metabolite in theeukaryotic cell in the absence of cyclodextrins but that detectionbecomes possible in the cell and the extracellular medium upon additionof cyclodextrins according to the disclosure. With an increase in theproduction of one or more sapogenins according to the method of thedisclosure, it is understood that production may be enhanced or inducedwith a factor 2, 3, 4, 5, 10, 20, 50, 100 or more, relative to theproduction in the absence of cyclodextrins.

Eukaryotic cells provided in the above-described method can be of anyunicellular or multicellular eukaryotic organism, but, in particularembodiments, microbial, plant, and algal cells are envisaged. The natureof the cells used will typically depend on the desired sapogenins and/orthe ease and cost of producing the sapogenins. According to particularembodiments, the plant cell as used is derived from a plant of the genusselected from the group comprising Medicago, Nova, Bupleurum, Maesa,Saponaria, Betula, Quillaja, Aesculus, Chenopodium, Hedera, Acacia,Centella, Oleander, Avena, Arabidopsis, or Nicotiana. The term “plant”as used herein refers to vascular plants (e.g., gymnosperms andangiosperms). According to further particular embodiments, the“microbial cell” as used herein is a yeast cell, in particular, a yeastcell of a Saccharomyces species (e.g., Saccharomyces cerevisiae), aHansenula species (e.g., Hansenula polymorpha), a Yarrowia species(e.g., Yarrowia lipolytica), a Kluyveromyces species (e.g.,Kluyveromyces lactis), a Pichia species (e.g., Pichia pastoris) or aCandida species (e.g., Candida utilis). According to a specificembodiment, the eukaryotic cells as used are Saccharomyces cells.According to further particular embodiments, the algal cells are derivedfrom algae of the genus selected from the group comprising Dunaliella,Chlorella, or Chlamydomonas. According to a very particular embodiment,the eukaryotic cells as used are not plant cells.

In a particular embodiment, the eukaryotic cells may naturally have thecapability of synthesizing triterpene saponins and triterpene sapogeninbuilding blocks, such as plant cells. A “plant cell” is understood,according to the disclosure, as being any cell that is derived from orfound in a plant and that is able to form or is part of undifferentiatedtissues, such as calli or cell cultures, differentiated tissues such asembryos, parts of plants, plants or seeds.

In an alternative embodiment, the “eukaryotic cells,” as used herein,themselves do not naturally produce triterpenoid sapo(ge)nins, but maydo so after genetic engineering. Thus, preferably, eukaryotic cellsartificially producing sapogenins refers to cells that, while notnaturally having the ability to synthesize sapogenins, have acquiredsuch ability by means of genetic manipulation processes includingtransgenesis. This particularly applies to yeast cells or algal cells,which naturally do not synthesize sapogenins. In yet another embodiment,the eukaryotic cells as used herein may be genetically engineered toproduce another spectrum of sapogenins, as compared to the naturalspectrum that is produced by the wild-type strain, which particularlymay apply to plant cells.

Thus, according to a preferred embodiment, the “plant cell,” as usedherein, may be a genetically engineered plant cell, which is a plantcell derived from a transgenic plant. A “transgenic plant,” as usedherein, refers to a plant comprising a recombinant polynucleotide and/ora recombinant polypeptide resulting in the expression of a regulatory orbiosynthetic enzyme of the sapogenin biosynthesis pathway. A transgenicplant refers to a whole plant as well as to a plant part, such as seed,fruit, leaf, or root, plant tissue, plant cells or any other plantmaterial, and progeny thereof. A transgenic plant can be obtained bytransforming a plant cell with an expression cassette and regeneratingsuch plant cell into a transgenic plant. Such plants can be propagatedvegetatively or reproductively. The transforming step may be carried outby any suitable means, including by Agrobacterium-mediatedtransformation and non-Agrobacterium-mediated transformation, asdiscussed further below. Plants can be regenerated from the transformedcell (or cells) by techniques known to those skilled in the art. Wherechimeric plants are produced by the process, plants in which all cellsare transformed may be regenerated from chimeric plants havingtransformed germ cells, as is known in the art. Methods that can be usedto transform plant cells or tissue with expression vectors include bothAgrobacterium and non Agrobacterium vectors. Agrobacterium-mediated genetransfer exploits the natural ability of Agrobacterium tumefaciens totransfer DNA into plant chromosomes and is described in detail in G.Gheysen, G. Angenon, and M. Van Montagu, 1998, Agrobacterium-mediatedplant transformation: a scientifically intriguing story with significantapplications in K. Lindsey (Ed.), Transgenic Plant Research, HarwoodAcademic Publishers, Amsterdam, pp. 1-33; and in H. A. Stafford (2000),Botanical Review 66:99-118. A second group of transformation methods isthe non-Agrobacterium-mediated transformation and these methods areknown as direct gene transfer methods. An overview is brought by P.Barcelo and P. A. Lazzeri (1998), Direct gene transfer: chemical,electrical and physical methods in K. Lindsey (Ed.), Transgenic PlantResearch, Harwood Academic Publishers, Amsterdam, pp. 35-55. Methodsinclude particle gun delivery, microinjection, electroporation of intactcells, polyethyleneglycol-mediated protoplast transformation,electroporation of protoplasts, liposome-mediated transformation,silicon-whiskers-mediated transformation, etc. A suitable control plantwould include a genetically unaltered or non-transgenic plant of theparental line (wild-type) used to generate a transgenic plant herein.

Genetically transformed hairy root cultures can be obtained bytransformation with virulent strains of Agrobacterium rhizogenes, andthey can produce high contents of secondary metabolites, includingtriterpenoid sapo(ge)nins, characteristic to the mother plant. Protocolsused for establishing of hairy root cultures vary, as well as thesusceptibility of plant species to infection by Agrobacterium (Toivounenet al. 1993; Vanhala et al. 1995). It is known that the Agrobacteriumstrain used for transformation has a great influence on root morphologyand the degree of secondary metabolite accumulation in hairy rootcultures. It is possible by systematic clone selection, e.g., viaprotoplasts, to find high yielding, stable, and from single-cell-derivedhairy root clones. This is possible because the hairy root culturespossess a great somaclonal variation. Another possibility oftransformation is the use of viral vectors (Turpen 1999).

Any plant tissue or plant cells capable of subsequent clonalpropagation, whether by organogenesis or embryogenesis, may betransformed with an expression vector of interest. The team“organogenesis” means a process by which shoots and roots are developedsequentially from meristematic centers; the term “embryogenesis” means aprocess by which shoots and roots develop together in a concertedfashion (not sequentially), whether from somatic cells or gametes. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include protoplasts, leaf disks,pollen, embryos, cotyledons, hypocotyls, megagametophytes, callustissue, existing meristematic tissue (e.g., apical meristems, axillarybuds, and root meristems), and induced meristem tissue (e.g., cotyledonmeristem and hypocotyls meristem).

According to a particularly preferred embodiment, the “yeast cell,” asused herein in any of the above-described methods, is a geneticallyengineered yeast cell, such as a yeast cell expressing an exogenousregulatory or biosynthetic enzyme of the sapogenin biosynthesis pathway(see also Tables 2 and 3). Preferably, the genetically engineered yeastcell is overexpressing an oxidosqualene cyclase (EC 5.4.99.-) and/or acytochrome P450 (EC 1.14.-). According to a particular embodiment, the“genetically engineered yeast cell,” as used herein in any of theabove-described methods, is overexpressing a cytochrome P450, which iscapable of hydroxylating a carbon at position 21 of an oleanane-typebackbone as pictured in FIG. 10. According to a preferred embodiment,the genetically engineered yeast cell as used in any of theabove-described methods is overexpressing SEQ ID NO:3 or 4, or fragmentsor variants thereof (as described further herein).

According to a further preferred embodiment, the genetically engineeredyeast cell as used is deficient in expression and/or activity of anenzyme involved in endogenous sterol synthesis. For example, and withoutthe purpose of being limitative, key enzymes in the yeast ergosterolbiosynthetic pathway may be down-regulated, in particular, thelanosterol synthase gene (EC 5.4.99.7) to enable maximal andcontrollable flux toward the heterologous production of the desiredtriterpenoid sapogenin compounds.

The term “endogenous” as used herein, refers to substances (e.g., genes)originating from within an organism, tissue, or cell. Analogously,“exogenous” as used herein, is any material originated outside of anorganism, tissue, or cell, but that is present (and typically can becomeactive) in that organism, tissue, or cell.

Table 2. Overview of oxidosqualene cyclase (OSCs) reported in theliterature as involved in sapogenin biosynthesis. 2,3-Oxidosqualenecyclization products identified to emerge from the activity of thecorresponding OSC are indicated in squared brackets: aa—α-amyrin,ba—β-amyrin, bau—baurenol, da—damyrin, dam—dammarenediol, fri—friedelin,ge—germanicol, glu—glutinol, isotir—isotirucallol, lu—lupeol,lud—lupane-3β,20-diol, tir—tirucalla-7,24-dien-3β-ol, minor—additionalbyproducts either reported to be of minor appearance or to represent<10% of the observed products (Table 2 derived from Augustin et al.2011).

Name GenBank ID Plant species Product Accurate b-amyrin synthases AaBASACA13386 A. annua [ba] AsOXA1 AAX14716 A. sedifolius [ba] AsbAS1CAC84558 A. strigosa [ba] BgbAS BAF80443 B. gymnorhiza [ba] BPY BAB83088B. platyphylla [ba] EtAS BAE43642 E. tirucalli [ba] GgbAS1 BAA89815 G.glabra [ba] GsAS1 ACO24697 G. straminea [ba] cOSC1 BAE53429 L. japonicus[ba] b-AS = MtAMY1 CAD23247 M. truncatula [ba] NsbAS1 ACH88048 N. sativa[ba] PNY BAA33461 P. ginseng [ba] PNY2 BAA33722 P. ginseng [ba] PSYBAA97558 P. sativum [ba] SlTTS1 ADU52574 S. lycopersicum [ba] SvBSABK76265 S. vaccaria [ba] Accurate lupeol synthases BgLUS BAF80444 B.gymnorhiza [lu] BPW BAB83087 B. platyphylla [lu] GgLUS1 BAD08587 G.glabra [lu] cOSC3 BAE53430 L. japonicus [lu] OEW BAA86930 O. europaea[lu] RcLUS ABB76766 R. communis [lu] TRW BAA86932 T. officinale [lu]Accurate dammarenediol synthases CaDDS AAS01523 C. asiatica [dam] PNA =DDS BAF33291 P. ginseng [dam] DDS = PNA ACZ71036 P. ginseng [dam]Multifunctional OSCs LUP1/At1g78970 NP_178018 A. thaliana [lu/lud + 4minor] LUP5/At1g66960 NP_176868 A. thaliana [tir/isotir + 4PEN6/At1g78500 NP_177971 A. thaliana [lu/bau/aa + 5 CsOSC2/CSV BAB83254C. speciosus [ba/ge/lu + add. LjAMY2 AAO33580 L. japonicus [ba/lu + 1minor] KcMS BAF35580 K. candel [lu/ba/aa] KdGLS ADK35124 K.daigremontiana [glu/fri/ba + 1 OEA BAF63702 O. europaea [aa/ba + 3minor]PSM BAA97559 P. sativum [aa/ba + 6minor] RsM1 BAF80441 R. stylosa[ge/ba + 1 minor] SlTTS2 ADU52575 S. lycopersicum [da/aa/ba + 4 minor]

TABLE 3 Overview of CYPs reported in the literature as involved insapogenin biosynthesis. Name GenBank ID Plant species Reference CYP51H10ABG88965 A. strigosa (Kunii et al., 2012) CYP716A12 FN995113 M.truncatula (Carelli et al., 2011) CYP716A15 BAJ84106 V. vinifera(Fukushima et al., 2011) CYP716A17 BAJ84107 V. vinifera (Fukushima etal., 2011) CYP716AL1 FN995113 C. roseus (Huang et al., 2012) CYP716A47AEY75212 P. ginseng (Han et al., 2011) CYP716A53v2 AFO63031 P. ginseng(Han et al., 2012) CYP72A61v2 BAL45199 M. truncatula (Fukushima et al.,2013) CYP72A63 AB558146 M. truncatula (Seki et al., 2011) CYP72A68v2BAL45204 M. truncatula (Fukushima et al., 2013) CYP72A154 AB558153 G.uralensis (Seki et al., 2011) CYP88D6 AB433179 G. uralensis (Seki etal., 2008) CYP93E1 NM_001249225 G. max (Shibuya et al., 2006) CYP93E2DQ335790 M. truncatula (Li et al., 2007) CYP93E3 AB437320 G. uralensis(Seki et al., 2008)

Suitable cell culture media for eukaryotic cells, in particular, plantcells and microbial cells, are known in the art. For plant cells,exemplary media include standard growth media, many of which arecommercially available (e.g., Sigma Chemical Co., St. Louis, Mo.).Examples include Schenk-Hildebrandt (SH) medium, Linsmaier-Skoog (LS)medium, Murashige and Skoog (MS) medium, Gamborg's B5 medium, Nitsch &Nitsch medium, White's medium, and other variations and supplements wellknown to those of skill in the art (see, e.g., Plant Cell Culture,Dixon, ed. IRL Press, Ltd. Oxford (1985); and George et al., PlantCulture Media, Vol. 1, Formulations and Uses Exegetics Ltd. Wilts, UK,(1987)). (See, e.g., Plant Cell Culture, Dixon, ed. IRL Press, Ltd.Oxford (1985); and George et al., Plant Culture Media, Vol. 1,Formulations and Uses Exegetics Ltd. Wilts, UK, (1987)).

For yeast cells, exemplary media include standard growth media, many ofwhich are commercially available (e.g., Clontech, Sigma Chemical Co.,St. Louis, Mo.). Examples include Yeast Extract Peptone Dextrose (YPD orYPED) medium, Yeast Extract Peptone Glycerol (YPG or YPEG) medium,Hartwell's complete (HC) medium, Synthetic complete (SC) medium, YeastNitrogen Base (YNB), and other variations and supplements well known tothose of skill in the art (see, Yeast Protocol Handbook, Clontech).

The incubation conditions (temperature, photoperiod, shaking,auxin/cytokine hormone ratio, promoter-inducing conditions,promoter-repressing conditions, etc.) will depend, among other factors,on the cells to be incubated and are standard techniques in the art. Ina particular embodiment, the current disclosure can be combined withother known methods to enhance the production and/or the secretion oftriterpenoid sapogenin production in eukaryotic cell cultures, forexample (1) by improvement of the cell culture conditions, (2) bymetabolic engineering, (3) by the addition of specific elicitors to thecell culture.

Preferably, the eukaryotic cell is induced before it produces secondarymetabolites such as triterpenoid sapogenins, meaning that the cellculture is stimulated by the addition of an external factor. Externalfactors include the application of heat, the application of cold, theaddition of acids, bases, metal ions, fungal membrane proteins, sugarsand the like. In the case of plants, it is demonstrated that betterproduction of plant secondary metabolites occurs via elicitation.Elicitors are compounds capable of inducing defense responses in plants.These are usually not found in intact plants but their biosynthesis isinduced after wounding or stress conditions. Commonly used elicitors arejasmonates, mainly jasmonic acid and its methyl ester, methyl jasmonate.Jasmonates are linoleic acid derivatives of the plasma membrane anddisplay a wide distribution in the plant kingdom. They were originallyclassified as growth inhibitors or promoters of senescence but now ithas become apparent that they have pleiotropic effects on plant growthand development. Jasmonates appear to regulate cell division, cellelongation and cell expansion and thereby stimulate organ or tissueformation. They are also involved in the signal transduction cascadesthat are activated by stress situations such as wounding, osmoticstress, desiccation and pathogen attack. Methyl jasmonate (MeJA) isknown to induce the accumulation of numerous defense-related secondarymetabolites through the induction of genes coding for the enzymesinvolved in the biosynthesis of these compounds in plants. Jasmonatescan modulate gene expression from the (post)transcriptional to the(post)translational level, both in a positive as well as in a negativeway. Genes that are up-regulated are, e.g., defense- and stress-relatedgenes (PR proteins and enzymes involved with the synthesis ofphytoalexins and other secondary metabolites), whereas the activity ofhousekeeping proteins and genes involved with photosynthetic carbonassimilation are down-regulated. For example: the biosynthesis ofphytoalexins and other secondary products in plants can also be boostedup by signal molecules derived from microorganisms or plants (such aspeptides, oligosaccharides, glycopeptides, salicylic acid and lipophilicsubstances), as well as by various abiotic elicitors like UV-light,heavy metals (Cu, VOSO4, Cd) and ethylene. The effect of any elicitor isdependent on a number of factors, such as the specificity of anelicitor, elicitor concentration, the duration of the treatment andgrowth stage of the culture.

A number of suitable culture media for callus induction and subsequentgrowth on aqueous or solidified media are known. Exemplary media includestandard growth media, many of which are commercially available (e.g.,Sigma Chemical Co., St. Louis, Mo.). Examples include Schenk-Hildebrandt(SH) medium, Linsmaier-Skoog (LS) medium, Murashige and Skoog (MS)medium, Gamborg's B5 medium, Nitsch & Nitsch medium, White's medium, andother variations and supplements well known to those of skill in the art

During or after the sapogenin production in the growth medium of aeukaryotic cell culture, according to any of the above describedmethods, the sapogenins can be extracted from the cells. According to apreferred embodiment, the sapogenins are extracted from the culturemedium wherein the sapogenins are secreted. Accordingly, the methods ofproducing sapogenins may optionally also comprise the step of extractingthe sapogenins from the culture medium. Eventually, the sapogenins mayalso be further purified. Means that may be employed to this end areknown to the skilled person. Generally, triterpenoid sapogenins can bemeasured intracellularly or in the extracellular space by methods knownin the art. Such methods comprise analysis by thin-layer chromatography,high-pressure liquid chromatography, capillary electrophoresis, gaschromatography combined with mass spectrometric detection,radioimmuno-assay (RIA) and enzyme immuno-assay (ELISA). For example,Medicago triterpene sapo(ge)nin content can be analyzed by reverse-phaseUPLC/ICR/FT-MS, and is also further illustrated in the Examples section.

In a further aspect, the disclosure also provides a eukaryotic cellgenetically engineered to synthesize sapogenins and/or pathwayintermediates. In particular, the genetically engineered cell is a yeastcell, for example a Saccharomyces, Schizosaccharomyces, Pichia,Yarrowia, Candida or Hansenula cell. According to a particularlypreferred embodiment, the yeast cell is genetically engineered toexpress an exogenous regulatory or biosynthetic enzyme of the sapogeninbiosynthesis pathway (see also Tables 2 and 3). Preferably, the yeastcell is genetically engineered to overexpress an oxidosqualene cyclase(EC 5.4.99.-) and/or a cytochrome P450 (EC 1.14.-). According to aparticular embodiment, the yeast cell is genetically engineered tooverexpress a cytochrome P450, which is capable of hydroxylating acarbon at position 21 of an oleanane-type backbone as pictured in FIG.10. According to a preferred embodiment, the yeast cell is geneticallyengineered to overexpress SEQ ID NO:3 or 4, or fragments or variantsthereof (as described further herein).

In still another aspect, the disclosure also encompasses existing ornovel sapogenins obtained by any of the above-described methods. Thesapogenins that are extracellularly accumulating in the growth medium ofa eukaryotic cell culture in the presence of cyclodextrins are readilyaccessible and can be exploited by industry for a variety of purposes,either directly, or after further synthetic chemistry. For example,sapogenins and its derivatives (including saponins) can be used asadditives to foods and cosmetics, preservatives, flavor modifiers,agents for removal of cholesterol from dietary products, and may also bevery valuable for their pharmacological properties. For example, severalsaponins and sapogenins are considered to possess activities such asanti-inflammatory, anti-carcinogenic, anti-bacterial, anti-fungal andantiviral effects. Saponins are also of interest as valuable adjuvantsand the first saponin-based vaccines are introduced commercially(reviewed in Sun et al. 2009).

In a specific embodiment, sapogenins that are hydroxylated on a carbonat position 21 of an oleanane-type backbone as pictured in FIG. 10 areencompassed here.

According to another aspect, the disclosure relates to an isolatedpolypeptide selected from the group consisting of:

-   -   (a) a polypeptide encoded by a polynucleotide comprising SEQ ID        NO:1 or 2;    -   (b) a polypeptide comprising a polypeptide sequence having at        least 75% identity to the polypeptide encoded by a        polynucleotide sequence having SEQ ID NO:1 or 2;    -   (c) a polypeptide comprising an amino acid sequence as set forth        in SEQ ID NO:3 or 4;    -   (d) a polypeptide comprising an amino acid sequence with at        least 75% identity to SEQ ID NO:3 or 4;    -   (e) fragments and/or variants of the polypeptides according to        (a), (b), (c), or (d).

In one embodiment, the disclosure relates to any of the above-describedpolypeptides wherein the polypeptide sequence is consisting of an aminoacid sequence as set forth in SEQ ID NO:3 or 4 and polypeptide sequenceshaving at least 75% identity to SEQ ID NO:3 or 4.

According to another aspect, the disclosure relates to an isolatedpolynucleotide selected from the group consisting of:

-   -   (a) a polynucleotide comprising a polynucleotide sequence having        the sequence SEQ ID NO:1 or 2;    -   (b) a polynucleotide comprising a polynucleotide sequence having        at least 70% identity to the sequence having SEQ ID NO:1 or 2;    -   (c) a polynucleotide that encodes the polypeptide sequence as        set forth in SEQ ID NO:3 or 4;    -   (d) a polynucleotide that encodes the polypeptide sequence as        set forth in SEQ ID NO:3 or 4;    -   (e) fragments and variants of the polynucleotides according to        (a), (b), (c) or (d).

As used herein, the terms “polypeptide,” “protein,” and “peptide” areused interchangeably and refer to a polymeric form of amino acids of anylength, which can include coded and non-coded amino acids, chemically orbiochemically modified or derivatized amino acids, and polypeptideshaving modified peptide backbones.

As used herein, the terms “nucleic acid,” “polynucleotide,” and“polynucleic acid” are used interchangeably and refer to a polymericform of nucleotides of any length, either deoxyribonucleotides orribonucleotides, or analogs thereof. Polynucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. Non-limiting examples of polynucleotides include a gene, a genefragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomalRNA, ribozymes, cDNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,control regions, isolated RNA of any sequence, nucleic acid probes, andprimers. The polynucleotide molecule may be linear or circular. Thepolynucleotide may comprise a promoter, an intron, an enhancer region, apolyadenylation site, a translation initiation site, 5′ or 3′untranslated regions, a reporter gene, a selectable marker or the like.The polynucleotide may comprise single-stranded or double-stranded DNAor RNA. The polynucleotide may comprise modified bases or a modifiedbackbone. A nucleic acid that is up to about 100 nucleotides in length,is often also referred to as an oligonucleotide.

An “isolated polypeptide” or an “isolated polynucleotide,” as usedherein, refers to, respectively, an amino acid sequence or apolynucleotide sequence that is not naturally occurring or no longeroccurring in the natural environment wherein it was originally present.

As used herein, the terms “identical” or “percent identity,” in thecontext of two or more nucleic acids or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame (e.g., 75% identity over a specified region), when compared andaligned for maximum correspondence over a comparison window ordesignated region as measured using sequence comparison algorithms or bymanual alignment and visual inspection. Preferably, the identity existsover a region that is at least about 25 amino acids or nucleotides inlength, or, more preferably, over a region that is 50-100 amino acids ornucleotides or even more in length. According to preferred embodiments,the disclosure relates to an isolated polypeptide comprising apolypeptide sequence having at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% identity to the polypeptide encoded by a polynucleotidesequence having SEQ ID NO:1 or 2, or having at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identity to a polypeptide comprising an aminoacid sequence as set forth in SEQ ID NO:3 or 4. In other preferredembodiments, the disclosure relates to an isolated polynucleotidecomprising a polynucleotide sequence having at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identity to the sequence havingSEQ ID NO:1 or 2.

In a particular embodiment, fragments and variants of any of the abovepolynucleotides or polypeptides also form part of this disclosure.

In reference to a nucleotide sequence, “a fragment” refers to anysequence of at least 15 consecutive nucleotides, preferably at least 30consecutive nucleotides, more preferably at least 50, 60, 70, 80, 90,100, 150, 200 consecutive nucleotides or more, of any of the sequencesprovided herein. If desired, the fragment may be fused at eitherterminus to additional base pairs, which may number from 1 to 20,typically 50 to 100, but up to 250 to 500 or more.

A “fragment,” as referred to polypeptides, refers to a subsequence ofthe polypeptide. Fragments may vary in size from as few as five aminoacids to the length of the intact polypeptide, but are preferably atleast 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75amino acids in length. If desired, the fragment may be fused at eitherterminus to additional amino acids, which may number from 1 to 20,typically 50 to 100, but up to 250 to 500 or more. A “functionalfragment” means a polypeptide fragment possessing the ability tohydroxylate a carbon at position 21 of an oleanane-type backbone aspictured in FIG. 10.

A “variant” as used herein refers to homologs, orthologs and paralogsand include, but are not limited to, homologs, orthologs and paralogs ofSEQ ID NOS:1-4. Homologs of a protein encompass peptides, oligopeptidesand polypeptides having amino acid substitutions, deletions and/orinsertions, preferably by a conservative change, relative to theunmodified protein in question and having similar biological andfunctional activity as the unmodified protein from which they arederived; or in other words, without significant loss of function oractivity. Orthologs and paralogs, which are well-known terms by theskilled person, define subcategories of homologs and encompassevolutionary concepts used to describe the ancestral relationships ofgenes. Paralogs are genes within the same species that have originatedthrough duplication of an ancestral gene; orthologs are genes fromdifferent organisms that have originated through speciation, and arealso derived from a common ancestral gene. Several different methods areknown by those of skill in the art for identifying and defining thesefunctionally homologous sequences. General methods for identifyingorthologues and paralogues include phylogenetic methods, sequencesimilarity and hybridization methods. Percentage similarity and identitycan be determined electronically. Examples of useful algorithms arePILEUP (Higgins & Sharp, CABIOS 5:151 (1989)), BLAST and BLAST 2.0(Altschul et al., J. Mol. Biol. 215:403 (1990); software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/)). Preferably,the homologue, orthologue or paralogue has a sequence identity at aprotein level of at least 50%, preferably 60%, more preferably 70%, evenmore preferably 80%, most preferably 90% as measured in a BLASTp.

Further, it will be appreciated by those of skill in the art, that anyof a variety of polynucleotide sequences are capable of encoding thepolypeptides of the disclosure. Due to the degeneracy of the geneticcode, many different polynucleotides can encode identical and/orsubstantially similar polypeptides. Sequence alterations that do notchange the amino acid sequence encoded by the polynucleotide are termed“silent” variations. With the exception of the codons ATG and TGG,encoding methionine and tryptophan, respectively, any of the possiblecodons for the same amino acid can be substituted by a variety oftechniques, e.g., site-directed mutagenesis, available in the art.Accordingly, any and all such variations of a sequence are a feature ofthe disclosure. In addition to silent variations, other conservativevariations that alter one, or a few amino acids in the encodedpolypeptide, can be made without altering the function of thepolypeptide (i.e., enhanced secondary metabolite production, in thecontext of the disclosure), these conservative variants are, likewise, afeature of the disclosure.

Conservative substitutions or variations, as used herein, are those inwhich at least one residue in the amino acid sequence has been removedand a different residue inserted in its place. Such substitutionsgenerally are made in accordance with the table depicted below. Thistable shows amino acids that can be substituted for an amino acid in aprotein and that are typically regarded as conservative substitutions.

Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp GluGln Asn Cys Ser Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, ValLys Arg, Gln Met Leu, Ile Phe Met, Leu, Tyr Ser Thr, Gly Thr Ser, ValTrp Tyr Tyr Trp, Phe Val Ile, Leu

Substitutions that are less conservative than those in the above tablecan be selected by picking residues that differ more significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. The substitutionsthat, in general, are expected to produce the greatest changes inprotein properties will be those in which (a) a hydrophilic residue,e.g., seryl or threonyl, is substituted for (or by) a hydrophobicresidue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) acysteine or proline is substituted for (or by) any other residue; (c) aresidue having an electropositive side chain, e.g., lysyl, arginyl, orhistidyl, is substituted for (or by) an electronegative residue, e.g.,glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine.

Substitutions, deletions and insertions introduced into the sequencesare also envisioned by the disclosure. Such sequence modifications canbe engineered into a sequence by site-directed mutagenesis or the othermethods known in the art. Amino acid substitutions are typically ofsingle residues; insertions usually will be on the order of from aboutone to ten amino acid residues; and deletions will range from about oneto thirty residues. In preferred embodiments, deletions or insertionsare made in adjacent pairs, e.g., a deletion of two residues orinsertion of two residues. Substitutions, deletions, insertions or anycombination thereof can be combined to arrive at a sequence. Themutations that are made in the polynucleotides of the disclosure shouldnot create complementary regions that could produce secondary mRNAstructure. Preferably, the polypeptide encoded by the DNA performs thedesired function (i.e., hydroxylating a carbon at position 21 of anoleanane-type backbone as pictured in FIG. 10, in the context of thisdisclosure).

In a specific embodiment, the disclosure relates to a chimeric genecomprising the following operably linked sequences: a) a promoter regioncapable of directing expression in a eukaryotic cell (as definedhereinbefore); b) a DNA region encoding a polypeptide as defined above;and c) a 3′ polyadenylation and transcript termination region.

The term “operably linked” as used herein refers to a linkage in whichthe regulatory sequence is contiguous with the gene of interest tocontrol the gene of interest, as well as regulatory sequences that actin trans or at a distance to control the gene of interest. For example,a DNA sequence is operably linked to a promoter when it is ligated tothe promoter downstream with respect to the transcription initiationsite of the promoter and allows transcription elongation to proceedthrough the DNA sequence. A DNA for a signal sequence is operably linkedto DNA coding for a polypeptide if it is expressed as a pre-protein thatparticipates in the transport of the polypeptide. Linkage of DNAsequences to regulatory sequences is typically accomplished by ligationat suitable restriction sites or adapters or linkers inserted in lieuthereof using restriction endonucleases known to one of skill in theart.

The term “regulatory sequence” as used herein refers to polynucleotidesequences that are necessary to affect the expression of codingsequences to which they are operably linked. Expression controlsequences are sequences that control the transcription,post-transcriptional events and translation of nucleic acid sequences.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism. The term “control sequences” isintended to include, at a minimum, all components whose presence isessential for expression, and can also include additional componentswhose presence is advantageous, for example, leader sequences and fusionpartner sequences.

A vector comprising a polynucleotide sequence or a chimeric gene asdefined above also forms part of the disclosure, as well as a host cellcomprising a polynucleotide sequence or a chimeric gene or a vector asdefined above.

The term “vector” as used herein is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid molecule to whichit has been linked. The vector may be of any suitable type including,but not limited to, a phage, virus, plasmid, phagemid, cosmid, bacmid oreven an artificial chromosome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g., vectorshaving an origin of replication that functions in the host cell). Othervectors can be integrated into the genome of a host cell uponintroduction into the host cell, and are thereby replicated along withthe host genome. Moreover, certain preferred vectors are capable ofdirecting the expression of certain genes of interest. Such vectors arereferred to herein as “recombinant expression vectors” (or simply,“expression vectors”). Suitable vectors have regulatory sequences, suchas promoters, enhancers, terminator sequences, and the like as desiredand according to a particular host organism (e.g., plant cell).Typically, a recombinant vector according to the disclosure comprises atleast one “chimeric gene” or “expression cassette.” Expression cassettesare generally DNA constructs preferably including (5′ to 3′ in thedirection of transcription): a promoter region, a polynucleotidesequence, homologue, variant or fragment thereof of the disclosureoperably linked with the transcription initiation region, and atermination sequence including a stop signal for RNA polymerase and apolyadenylation signal. It is understood that all of these regionsshould be capable of operating in biological cells, such as plant cells,to be transformed. The promoter region comprising the transcriptioninitiation region, which preferably includes the RNA polymerase bindingsite, and the polyadenylation signal may be native to the biologicalcell to be transformed or may be derived from an alternative source,where the region is functional in the biological cell.

The term “recombinant host cell” (“expression host cell,” “expressionhost system,” “expression system” or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinant vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. A recombinant host cell may be an isolated cell or cellline grown in culture or may be a cell that resides in a living tissueor organism. Host cells can be of bacterial, fungal, plant or mammalianorigin.

According to yet another aspect, the disclosure provides a transgenicplant or a cell derived thereof that is transformed with theabove-described vector.

The term “plant” as used herein refers to vascular plants (e.g.,gymnosperms and angiosperms). A “transgenic plant” refers to a plantcomprising a recombinant polynucleotide and/or a recombinant polypeptideaccording to the disclosure. A transgenic plant refers to a whole plantas well as to a plant part, such as seed, fruit, leaf, or root, planttissue, plant cells or any other plant material, and progeny thereof. Atransgenic plant can be obtained by transforming a plant cell with anexpression cassette of the disclosure and regenerating such plant cellinto a transgenic plant. Such plants can be propagated vegetatively orreproductively. The transforming step may be carried out by any suitablemeans, including by Agrobacterium-mediated transformation andnon-Agrobacterium-mediated transformation, as discussed in detail below.Plants can be regenerated from the transformed cell (or cells) bytechniques known to those skilled in the art. Where chimeric plants areproduced by the process, plants in which all cells are transformed maybe regenerated from chimeric plants having transformed germ cells, as isknown in the art. Methods that can be used to transform plant cells ortissue with expression vectors of the disclosure include bothAgrobacterium and non-Agrobacterium vectors. Agrobacterium-mediated genetransfer exploits the natural ability of Agrobacterium tumefaciens totransfer DNA into plant chromosomes and is described in detail in G.Gheysen, G. Angenon, and M. Van Montagu, 1998, Agrobacterium-mediatedplant transformation: a scientifically intriguing story with significantapplications in K. Lindsey (Ed.), Transgenic Plant Research, HarwoodAcademic Publishers, Amsterdam, pp. 1-33; and in H. A. Stafford (2000),Botanical Review 66:99-118. A second group of transformation methods isthe non-Agrobacterium-mediated transformation and these methods areknown as direct gene transfer methods. An overview is brought by P.Barcelo and P. A. Lazzeri (1998), Direct gene transfer: chemical,electrical and physical methods in K. Lindsey (Ed.), Transgenic PlantResearch, Harwood Academic Publishers, Amsterdam, pp. 35-55.

Methods include particle gun delivery, microinjection, electroporationof intact cells, polyethyleneglycol-mediated protoplast transformation,electroporation of protoplasts, liposome-mediated transformation,silicon-whiskers-mediated transformation, etc.

Hairy root cultures can be obtained by transformation with virulentstrains of Agrobacterium rhizogenes, and they can produce high contentof secondary metabolites characteristic to the mother plant. Protocolsused for establishing of hairy root cultures vary, as well as thesusceptibility of plant species to infection by Agrobacterium (Toivounenet al. 1993; Vanhala et al. 1995). It is known that the Agrobacteriumstrain used for transformation has a great influence on root morphologyand the degree of secondary metabolite accumulation in hairy rootcultures. It is possible by systematic clone selection, e.g., viaprotoplasts, to find high yielding, stable, and from single-cell-derivedhairy root clones. This is possible because the hairy root culturespossess a great somaclonal variation. Another possibility oftransformation is the use of viral vectors (Turpen 1999).

Any plant tissue or plant cells capable of subsequent clonalpropagation, whether by organogenesis or embryogenesis, may betransformed with an expression vector of the disclosure. The term“organogenesis” means a process by which shoots and roots are developedsequentially from meristematic centers; the term “embryogenesis” means aprocess by which shoots and roots develop together in a concertedfashion (not sequentially), whether from somatic cells or gametes. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include protoplasts, leaf disks,pollen, embryos, cotyledons, hypocotyls, megagametophytes, callustissue, existing meristematic tissue (e.g., apical meristems, axillarybuds, and root meristems), and induced meristem tissue (e.g., cotyledonmeristem and hypocotyls meristem).

A “control plant” as used in the disclosure refers to a plant cell,seed, plant component, plant tissue, plant organ or whole plant used tocompare against transgenic or genetically modified plant for the purposeof identifying a difference in production of sapogenins in thetransgenic or genetically modified plant. A control plant may in somecases be a transgenic plant line that comprises an empty vector ormarker gene, but does not contain the recombinant polynucleotide of thedisclosure that is expressed in the transgenic or genetically modifiedplant being evaluated. In general, a control plant is a plant of thesame line or variety as the transgenic or genetically modified plantbeing tested. A suitable control plant would include a geneticallyunaltered or non-transgenic plant of the parental line (wild-type) usedto generate a transgenic plant herein.

Plants of the disclosure may include, but are not limited to, plants orplant cells of agronomically important crops that are or are notintended for animal or human nutrition, such as maize or corn, wheat,barley, oat, Brassica spp. plants such as Brassica napus or Brassicajuncea, soybean, bean, alfalfa, pea, rice, sugarcane, beetroot, tobacco,sunflower, cotton, Arabidopsis, vegetable plants such as cucumber, leek,carrot, tomato, lettuce, peppers, melon, watermelon, diverse herbs suchas oregano, basilicum and mint. It may also be applied to plants thatproduce valuable compounds, e.g., useful as, for instance,pharmaceuticals, as ajmalicine, vinblastine, vincristine, ajmaline,reserpine, rescinnamine, camptothecine, ellipticine, quinine, andquinidine, taxol, morphine, scopolamine, atropine, cocaine,sanguinarine, codeine, genistein, daidzein, digoxinu, calystegins or asfood additives such as anthocyanins, and vanillin, including, but notlimited to, the classes of compounds mentioned above. Examples of suchplants include, but not limited to, Papaver spp., Rauwolfia spp., Taxusspp., Cinchona spp., Eschscholtzia californica, Camptotheca acuminata,Hyoscyamus spp., Berberis spp., Coptis spp., Datura spp., Atropa spp.,Thalictrum spp., Peganum spp. Preferred members of the genus Taxuscomprise Taxus brevifolia, Taxus baccata, Taxus cuspidata, Taxuscanadensis and Taxus floridana.

The polynucleotide sequence, homologue, variant or fragment thereof ofthe disclosure may be expressed in, for example, a plant cell under thecontrol of a promoter that directs constitutive expression or regulatedexpression. Regulated expression comprises temporally or spatiallyregulated expression and any other form of inducible or repressibleexpression. Temporally means that the expression is induced at a certaintime point, for instance, when a certain growth rate of the plant cellculture is obtained (e.g., the promoter is induced only in thestationary phase or at a certain stage of development). Spatially meansthat the promoter is only active in specific organs, tissues, or cells(e.g., only in roots, leaves, epidermis, guard cells or the like). Otherexamples of regulated expression comprise promoters whose activity isinduced or repressed by adding chemical or physical stimuli to the plantcell. In a preferred embodiment, the expression is under control ofenvironmental, hormonal, chemical, and/or developmental signals. Suchpromoters for plant cells include promoters that are regulated by (1)heat, (2) light, (3) hormones, such as abscisic acid, and methyljasmonate (4) wounding or (5) chemicals such as salicylic acid,chitosans or metals. Indeed, it is well known that the expression ofsecondary metabolites can be boosted by the addition of, for example,specific chemicals, jasmonate and elicitors. In a particular embodiment,the co-expression of several (more than one) polynucleotide sequences orhomologues or variants or fragments thereof, in combination with theinduction of secondary metabolite synthesis, is beneficial for anoptimal and enhanced production of secondary metabolites. Alternatively,the at least one polynucleotide sequence, homologue, variant or fragmentthereof is placed under the control of a constitutive promoter. Aconstitutive promoter directs expression in a wide range of cells undera wide range of conditions. Examples of constitutive plant promotersuseful for expressing heterologous polypeptides in plant cells include,but are not limited to, the cauliflower mosaic virus (CaMV) 35Spromoter, which confers constitutive, high-level expression in mostplant tissues including monocots, the nopaline synthase promoter and theoctopine synthase promoter. The expression cassette is usually providedin a DNA or RNA construct that is typically called an “expressionvector,” which is any genetic element, e.g., a plasmid, a chromosome, avirus, behaving either as an autonomous unit of polynucleotidereplication within a cell (i.e., capable of replication under its owncontrol) or being rendered capable of replication by insertion into ahost cell chromosome, having attached to it another polynucleotidesegment, so as to bring about the replication and/or expression of theattached segment. Suitable vectors include, but are not limited to,plasmids, bacteriophages, cosmids, plant viruses and artificialchromosomes. The expression cassette may be provided in a DNA construct,which also has at least one replication system. In addition to thereplication system, there will frequently be at least one markerpresent, which may be useful in one or more hosts, or different markersfor individual hosts. The markers may a) code for protection against abiocide, such as antibiotics, toxins, heavy metals, certain sugars orthe like; b) provide complementation, by imparting prototrophy to anauxotrophic host; or c) provide a visible phenotype through theproduction of a novel compound in the plant. Exemplary genes that may beemployed include neomycin phosphotransferase (NPTII), hygromycinphosphotransferase (HPT), chloramphenicol acetyltransferase (CAT),nitrilase, and the gentamicin resistance gene. For plant host selection,non-limiting examples of suitable markers are β-glucuronidase, providingindigo production, luciferase, providing visible light production, GreenFluorescent Protein and variants thereof, NPTII, providing kanamycinresistance or G418 resistance, HPT, providing hygromycin resistance, andthe mutated aroA gene, providing glyphosate resistance.

The term “promoter activity” refers to the extent of transcription of apolynucleotide sequence, homologue, variant or fragment thereof that isoperably linked to the promoter whose promoter activity is beingmeasured. The promoter activity may be measured directly by measuringthe amount of RNA transcript produced, for example, by Northern blot orindirectly by measuring the product coded for by the RNA transcript,such as when a reporter gene is linked to the promoter.

According to a further aspect of the disclosure, the above-describedpolynucleotide sequences (and encoded proteins) can be used for thebiosynthesis of (novel) triterpenoid sapogenin compounds. To illustratethis further, without the purpose of being limitative, one is referredto the Examples section below.

The following examples are intended to promote a further understandingof the disclosure. While this disclosure is described herein withreference to illustrated embodiments, it should be understood that thedisclosure is not limited hereto. Those having ordinary skill in the artand access to the teachings herein will recognize additionalmodifications and embodiments within the scope thereof. Therefore, thedisclosure is limited only by the claims attached herein.

EXAMPLES I. Introduction Example 1. Triterpenoid Sapogenins in PlantCultures

In an attempt to detect triterpenoid sapogenins from plant cultures,hairy roots of the model legume Medicago truncatula transformed with aGATEWAY™ plasmid pK7WG2D-GUS, containing a non-functionalβ-glucuronidase gene (GUS) expressed from a ³⁵S promoter (Pollier etal., 2011) were analyzed. The hairy roots were grown for three weeks in30 ml Murashige and Skoog basal salt mixture including vitamins(Duchefa) prior to an organic extraction and gas chromatography-massspectrometry (GC-MS) analysis. The roots were harvested from the culturemedium, frozen in liquid nitrogen and ground to a fine powder. A totalmetabolite extraction was performed on this ground material using 1 mlof methanol. The methanol phase was evaporated to dryness and thesubsequent pellet was extracted with 1 ml hexane, since the triterpenoidsapogenins are extremely hydrophobic in nature. The hexane phase wasseparated from the undissolved residue, evaporated to dryness andtrimethylsilylated as described (Radosevich et al. 1985). Thisderivatized material was subjected to GC-MS (GC model 6890, MS model5973, Agilent) analysis where, a 1 μl aliquot was injected in splitlessmode into a VF-5 ms capillary column (VARIAN® CP9013, Agilent) andoperated at a constant helium flow of 1 ml/minute. The injectortemperature was set to 280° C. and the oven temperature was held at 80°C. for 1 minute post-injection, ramped to 280° C. at 20° C./minute, heldat 280° C. for 45 minutes, ramped to 320° C. at 20° C./minute, held at320° C. for 1 minute, and finally cooled down to 80° C. at 50° C./minuteat the end of the run. The MS transfer line was set to 250° C., the MSion source to 230° C., and the quadrupole to 150° C., throughout.

Owing to the hydrophobicity of sterols, multiple sterols and sterolintermediates were detected in the GC chromatogram of this extract (FIG.2). The identity of these compounds was confirmed using the MS electronionization (EI) pattern described in literature (data not shown).However, only trace amounts of erythrodiol and no other triterpenoidsapogenins were detected in this chromatogram (FIG. 2), emphasizingtheir low abundance in a glycosyl-free form in the hairy roots. Due tothis inability to detect triterpene sapogenins from plant cultures, ayeast strain capable of accumulating detectable amounts of thesevaluable compounds were engineered.

II. Production of Triterpenoid Sapogenins in Microbial Cultures Example2. Generation of Yeast Strain TM1 with Modified Sterol Biosynthesis

Triterpene saponin and sterol biosynthesis depend on the same precursor,i.e., oxidosqualene (FIG. 1). To enable maximal and controllable fluxtoward the heterologous production of the desired triterpene compounds,the endogenous sterol synthesis of the model yeast Saccharomycescerevisiae was first engineered, which leads to ergosterol as a majorcompound.

The ergosterol biosynthetic pathway of the Saccharomyces cerevisiaestrain S288c BY4742 was modified as described (Kirby et al., 2008) withadaptations. The lanosterol synthase gene (ERG7) (GenBank accessionnumber NM_001179202) was made conditionally down-regulatable byreplacing the native ERG7 promoter with a methionine-repressible MET3promoter as described for ERG9 in S. cerevisiae CEN.PK 113-7D(Asadollahi et al., 2008), to generate strain TM1. The amount ofergosterol produced by TM1 in the presence of different concentrationsof methionine was quantified using the sterol quantification method asdescribed (Arthington-Skaggs et al., 1999). A 60% reduction inergosterol accumulation was observed in TM1 with 1.5 mM methionine whencompared to wild-type cells. Further, a truncated, feedback-uncoupled,copy of isoform 1 of the rate-limiting enzyme3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1) (GenBank accessionnumber NM_001182434) was generated as described (Polakowski et al.,1998). The tHMG1 was cloned into the multiple cloning site (MCS) 1 ofthe high copy number plasmid pESC-URA (Agilent Technologies) behind thegalactose inducible GAL10 promoter to generate pESC-URA[GAL10/tHMG1],which was transformed into TM1 to generate strain TM5.

Example 3. Generation of β-Amyrin-Producing Yeast Strain TM3

The Glycyrrhiza glabra β-amyrin synthase (GgbAS) (GenBank accessionnumber AB037203; (Hayashi et al., 2001)) was cloned into the MCS 2 ofplasmid pESC-URA[GAL10/tHMG1] to generate pESC-URA[GAL10/tHMG1;GAL1/bAS]. Strain TM1 was transformed with this plasmid using thelithium acetate-mediated transformation method to generate strain TM3.To validate the production of β-amyrin in TM3, strains TM3 and TM5 werefirst precultured in minimal synthetic defined (SD) base-containing dropout (DO) supplement—Ura (SD-Ura) (Clontech) medium for 18-20 hours at30° C. and 250 rpm. The precultures were washed prior to inoculatingMinimal SD Base Gal/Raf containing DO supplement—Ura (SD Gal/Raf-Ura)(Clontech) medium to a starting optical density of 0.25. The cultureswere incubated as before for 24 hours, prior to addition of 10 mMmethionine to a final concentration of 1.5 mM, following which they wereincubated further for 48 hours. Yeast cells from a 1 ml culture wereused for extraction and gas chromatography-mass spectrometry (GC-MS)analysis as described (Kirby et al., 2008) with modifications. The cellpellet was resuspended in an equal volume of 40% potassium hydroxide and50% ethanol prior to lysis by boiling at 95° C. for 10 minutes. Anorganic extraction was performed on the lysate using an equivalentvolume of hexane and vortexing at high speed for 1 minute. The hexaneextraction was repeated thrice before the phases were pooled andevaporated to dryness. A trimethylsilyl derivatization was performed onthe dried material, and used for GC-MS analysis. The GC chromatogramsshowed the presence of a single peak at 27.2 minutes corresponding to36.2 mg/L of β-amyrin in TM3 but not in TM5 (FIG. 3). The E1 pattern forthis peak was identical to a standard of β-amyrin (Extrasynthese) (datanot shown). However, when a similar extraction and GC-MS analysis wasperformed on 1 ml of spent medium without yeast cells, no β-amyrin couldbe detected from either TM3 or TM5 (FIG. 3). In conclusion, a yeaststrain was engineered that is capable of synthesizing β-amyrin insignificant amounts; however, the β-amyrin that is generated is notsecreted to the extracellular medium.

Example 4. Generation of Lupeol-Producing Yeast Strain TM6

The Arabidopsis thaliana lupeol synthase (AtLUS1) (GenBank accessionnumber U49919; (Herrera et al., 1998)) was cloned into the MCS 2 ofplasmid pESC-URA[GAL10/tHMG1] to generate pESC-URA[GAL10/tHMG1;GAL1/AtLUS1]. The plasmid was transformed into strain TM1 to generate alupeol-producing strain TM6. The production of lupeol by strain TM6 wasverified by culturing as described in Example 3 and analyzing thetrimethylsilylated fraction by GC-MS. A single peak at 28.9 minutescorresponding to 46.3 mg/L of lupeol with an E1 pattern identical to astandard of lupeol (Extrasynthese) (data not shown), was observed in theGC chromatograms of TM6 but not TM5. Again, no lupeol was detected inthe spent medium of strains TM6 and TM5 (FIG. 4). It can, thus,similarly be concluded that, although lupeol is synthesized by theengineered yeast strain, it is not secreted to the growth medium.

Example 5. Cyclodextrin Facilitates Secretion of β-Amyrin and Lupeolinto the Medium

Strains TM3, TM6 and TM5 were cultured as in Example 2 withmodifications. The precultures were prepared and inoculated into SDGal/Raf-Ura medium and incubated for 24 hours as described. Along withmethionine, 250 mM methyl-β-cyclodextrin (CAVASOL©, Wacker QuimicaIbérica S.A.) was also added to a final concentration of 5 mM and thecultures were incubated further under the same conditions. Post 24 hoursincubation, 250 mM methyl-β-cyclodextrin was added once again to aconcentration of 5 mM and the cultures were incubated further for 24hours. The cells and spent medium were harvested from 1 ml of theculture and processed separately for extraction and GC-MS analysis asdescribed in Example 3. GC chromatograms confirmed the presence ofβ-amyrin and lupeol in the cell pellet as well as in spent medium ofstrains TM3 and TM6, respectively, but not TM5, with higherconcentrations of both β-amyrin and lupeol quantified from the extractsof spent medium (37.3 mg/L and 164.2 mg/L, respectively) when comparedto cell pellet (20.4 mg/L and 41.7 mg/L, respectively) (FIG. 5).Additionally, the total concentration of both β-amyrin and lupeol isfound to be 1.6-fold and 4.4-fold, respectively, higher in culturestreated with cyclodextrin when compared to non-treated controls. Theabsence of β-amyrin and lupeol in the extracts obtained from spentmedium of cells cultured in the absence of cyclodextrin and vice versastrongly underscore the effects of cyclodextrins on the secretion oftriterpene sapogenin backbones into the medium.

Further, it was determined whether cyclodextrin facilitates thesecretion of triterpene sapogenin backbones in a dose-dependent manner.For this, the β-amyrin-producing strain TM3 was employed and appliedcyclodextrin at different times during culturing. A total of sevenculturing conditions were set up, which included an untreated control(C) and six treated samples, with cyclodextrin added to a concentrationof 5 mM each time. To samples 11, 12 and 13, cyclodextrin was added onDay 1 immediately after inoculation into SD Gal/Raf-Ura medium. Tosamples I2 and I3, an additional dose of cyclodextrin was added on Day2, together with addition of methionine. Additionally, to sample I3, athird dose of cyclodextrin was added on Day 3. Further, to samples R1,R2, and AR1, cyclodextrin was added on; Day 2 only, Day 2 and Day 3, andDay 3 only, respectively. Extractions were performed on the spent mediumof all the samples on Day 4 and quantified for β-amyrin using GC-MS asdescribed. Surprisingly, a direct correlation was observed between theamount of β-amyrin quantified from the spent medium and the number oftimes cyclodextrin was added to the sample, thereby suggesting thedose-dependent nature of this secretion (FIG. 6). For the purpose of thefollowing experiments, being the generation of sapogenins derived fromits precursors (e.g., β-amyrin, lupeol), it was decided to employcondition R2 for all subsequent experiments. In this way, the excessivesecretion from the cells into the medium and, hence, loss of β-amyrin,which is the precursor for the consecutive cytochrome P450monooxygenases (CYPs), is prevented.

Example 6. Secretion of Triterpene Sapogenins from Strains TM10, TM11,TM12 and TM22

To produce triterpene sapogenins in the yeast strain, β-amyrin andlupeol were modified with three characterized cytochrome P450monooxygenases (CYPs), CYP716A12 (GenBank accession number FN995113;(Carelli et al., 2011)), CYP88D6 (GenBank accession number AB433179;(Seki et al., 2008)) and CYP93E2 (GenBank accession number DQ335790; (Liet al., 2007)). The CYPs need a CYP reductase (CPR) as a redox partnerfor their activity. Therefore, A. thaliana CPR, ATR1 (At4g24520) alongwith the CYPs were simultaneously cloned. Both the CYPs and CPR were PCRamplified and cloned into the entry vector pDONR221 by GATEWAY™recombination (Invitrogen Life Technologies). Further, the CYPs wereGATEWAY™ recombined into the high copy number expression vector,pAG423GAL-ccdB (Addgene plasmid 14149) containing the GAL1 promoter andHIS3 auxotrophic marker to generate the plasmids pAG423[GAL1/CYP716A12],pAG423[GAL1/CYP88D6] and pAG423[GAL1/CYP93E2]. The CPR was GATEWAY™recombined into the high copy number expression vector pAG425GAL-ccdB(Addgene plasmid 14153) having the GAL1 promoter and LEU2 auxotrophicmarker to generate plasmid pAG425[GAL1/AtATR1].

The TM3 strain was transformed with the plasmid pAG425[GAL1/AtATR1], incombination with either pAG423[GAL1/CYP716A12], pAG423[GAL1/CYP88D6],pAG423[GAL1/CYP93E2] or pAG423GAL-ccdB to generate strains TM10, TM11,TM12 and TM26, respectively. The spent medium of strains TM10, TM11,TM12 and TM26, cultured in SD Gal/Raf-Ura/-His/-Leu medium withcyclodextrin treatment as described in Example 5, was analyzed by GC-MS.Peaks corresponding to erythrodiol, oleanolic aldehyde and oleanolicacid at high concentrations were detected in the GC chromatograms ofextract from TM10, indicating the C-28 hydroxylation of β-amyrinmediated by CYP716A12. Similarly, 11-hydroxy-β-amyrin and11-oxo-β-amyrin were detected in the chromatogram of strain TM11 and24-hydroxy-β-amyrin in the extract of strain TM12. The identity of allthe hydroxylated β-amyrin peaks was confirmed by comparing their E1patterns against available standards (erythrodiol, oleanolic acid(Extrasynthese)), or previous reports when a commercial standard wasunavailable. No hydroxylated β-amyrin was detected in the chromatogramof TM26 (FIG. 7). The presence of triterpene sapogenins in the growthmedium of strains TM10, TM11 and TM12 suggest the role of cyclodextrinin the secretion of oleanane-type sapogenins from the yeast cells intothe culture medium.

Next, it was determined whether cyclodextrin could also facilitate thesecretion of lupane-type triterpene sapogenins from the cells to theculture medium. For this, strains TM22 and TM28 were generated bytransforming strain TM6 with the plasmids pAG425[GAL1/AtATR1] andpAG423[GAL1/CYP716A12] or pAG423GAL-ccdB, respectively. Strains TM22 andTM28 were cultured as described using SD Gal/Raf-Ura/-His/-Leu mediumwith cyclodextrin treatment and the spent medium was used for extractionand GC-MS analysis. 7.2 mg/L betulin and 2.4 mg/L betulinic acid weredetected in the chromatograms of TM22 but not TM28, indicating the C-28hydroxylation of lupeol by CYP716A12 (FIG. 8). The detection ofhydroxylated and carboxylated lupeol in the growth medium furthersupports the effect of cyclodextrin in the secretion of lupane-typesapogenins as well.

III. Production of Triterpenoid Sapogenins in Plant Cultures Example 7.Cyclodextrin Facilitates Secretion of Triterpene Sapogenins fromMedicago truncatula

It was then determined whether cyclodextrins could also facilitate theproduction and/or secretion of triterpene sapogenins from the modellegume M. truncatula by analyzing the spent medium of M. truncatulahairy roots transformed with pK7WG2D-GUS. The hairy roots were grown fortwo weeks in 20 ml Murashige and Skoog basal salt mixture includingvitamins (Duchefa) prior to addition of 250 mM cyclodextrin to a finalconcentration of 25 mM, in combination with or without 100 μM methyljasmonate, and compared to untreated and 100 μM methyl jasmonate onlytreated controls. The roots were harvested 48 hours after treatment andthe spent medium was extracted and analyzed by GC-MS. Surprisingly, thetriterpene sapogenins erythrodiol, oleanolic acid and oleanolicaldehyde, corresponding to the building blocks of the most abundantsaponins produced by the hairy roots (Pollier et al., 2011), could nowbe detected in the GC chromatograms of medium of roots treated withcyclodextrin, but not in control roots (FIG. 9). EI patterns werecompared against available standards to confirm the identity oferythrodiol and oleanolic acid (Extrasynthese), and previous reports foroleanolic aldehyde. Additionally, a significant increase, of up to150-fold for erythrodiol and 2-fold for oleanolic acid, was noted whencyclodextrin was combined with methyl jasmonate treatment (FIG. 9). Thisconfirms the role of cyclodextrins in the secretion of scarcelyintracellularly accumulating saponin intermediates, the sapogenins, fromplant cultures into the culturing medium, as described in Example 1.

IV. Identification and Characterization of Novel Saponin BiosyntheticGenes Example 8. Transcript Profiling of MeJA-Treated Bupleurum FalcatumReveals a Novel Plant Cytochrome P450

The genus Bupleurum consists of perennial herbs and forms an integralpart of Asian traditional medicine in which it is used, either alone orin combination with other ingredients, for the treatment of commoncolds, fever and inflammatory disorders in the form of over-the-counterherbal teas. Saikosaponins constitute the largest class of secondarymetabolites in Bupleurum and account for ˜7% of the total dry weight ofroots. More than 120 closely related glycosylated oleanane- andursane-type saikosaponins have been identified from this genus that canbe distinguished only by the positions and numbers of double bonds inrings C and D and oxygenation patterns on C-16, C-23, C-28 and C-30(FIG. 10) (Ashour and Wink, 2011). The presence of oxygenations atvarious positions on saikosapogenins suggests the presence of specificenzymes, generally CytP450s, capable of catalyzing these modificationson the β-amyrin and/or α-amyrin backbone in the genus Bupleurum.However, to date, not a single CytP450 or oxido-reductase involved intriterpene sapogenin biosynthesis has been identified from Bupleurumspecies.

To identify new saponin biosynthesis genes, a genome-widecDNA-AFLP®-based transcript profiling was performed on the roots ofhydroponically grown B. falcatum plants. B. falcatum seeds, obtainedfrom a commercial source (on the World Wide Web atSandMountainHerbs.com), were sown in soil, and 2 weeks aftergermination, seedlings were transferred to aerated hydroponics mediumcontaining 1 g/L 10-30-20 salts (Scotts, Ohio, USA), pH 6.5. Plants weregrown at 16 hours/8 hours light/dark regime, at 21° C. The pH wasmonitored daily and adjusted to 6.5 by adding KOH to the hydroponicsmedium. Three weeks after the plants were transferred to the hydroponicsmedium, they were treated with 50 μM methyl jasmonate (MeJA) (dissolvedin ethanol (EtOH)) or an equivalent amount of EtOH as a control, byadding the EtOH or MeJA solution directly to the hydroponics medium. Fortranscript profiling, roots were harvested 0, 0.5, 1, 2, 4, 8 and 24hours after treatment, frozen in liquid nitrogen, and stored at −70° C.For each sample, three individual plants were pooled.

A full genome-wide cDNA-AFLP®-based transcript profiling on the roots ofhydroponically grown B. falcatum plants was carried out as described inVuylsteke et al. (2007). Gel images were analyzed with the AFLP-QUANTAR®PRO software (KEYGENE®, Wageningen, The Netherlands), allowing accuratequantification of band intensities. Extraction and analysis ofexpression data of all individual bands, selection of tags displayingdifferential expression, cluster analysis, sequencing, and BLASTanalysis was performed as described (Rischer et al., 2006).

Using the complete set of 128 BstYI+1/MseI+2 primer combinations, theexpression of a total of 18,800 transcript tags was monitored over time.In total, 1,771 MeJA-responsive transcript tags were isolated (hereafterreferred to as “BF tags”). Direct sequencing of the reamplified BF tagsgave good-quality sequences for 1217 (68.7%) of the fragments. To theremaining 554 tags (31.3%), no unique sequence could be attributedunambiguously, indicating that they might not represent unique genetags, hence, these tags were not considered for further analysis. ABLAST search with the nucleotide sequences of the 1217 unique cDNA-AFLP®tags led to the annotation of 776 (63.7%) of the BF tags.

Average linkage hierarchical clustering analysis of the expressionprofiles of the 776 annotated BF tags showed that, upon MeJA treatment,the selected genes are either transcriptionally activated ortranscriptionally repressed. The activated gene tags can be divided intodifferent subclusters, based on the timing of the MeJA response. In onesubcluster, genes are activated within 2 hours after the MeJA treatment,and their expression remains high thereafter. In this group, tagscorresponding to genes encoding enzymes that catalyze early steps in thetriterpene saponin biosynthesis, including squalene synthase (SQS) andβ-amyrin synthase (bAS), can be found. These tags displayed an almostidentical expression pattern, suggesting a tight co-regulation, andreached maximum levels of expression 8-24 hours post-elicitation (FIG.11). The gene tag BF567 (hereafter named CYP716AO21) is tightlyco-regulated with these genes (FIG. 11), and shows homology to the M.truncatula gene encoding the cytochrome P450 enzyme CYP716A12 that wasrecently shown to oxidize β-amyrin in a sequential three-step oxidationon C-28 to yield oleanolic acid through erythrodiol (Carelli et al.,2011; Fukushima et al., 2011). The full-length open reading frame(BF567, hereafter named CYP716AO21) corresponding to the gene tagCYP716AO21 was picked up from a B. falcatum Uncut Nanoquantity cDNAlibrary (Pollier et al., 2011b). Using the primers (sense,5′-CCTCCTTATACATTCGTTCCATTC-3′ (SEQ ID NO:20) and antisense,5′-TTAGGGTCTACTTTCTCCCATTTG-3′ (SEQ ID NO:21), the full-length codingsequence of CYP716AO21 (SEQ ID NO:1) corresponding to the gene tagCYP716AO21 was picked up from the screening of a B. falcatum UncutNanoquantity cDNA library (custom-made by Invitrogen, Carlsbad, Calif.,USA) as reported (Pollier et al., 2011b). The full-length open readingframe (FL-ORF) of CYP716AO21 was PCR amplified for GATEWAY™ cloning intothe entry vector pDONR221 using the primer pair P19(GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGAACTTTCTATCACT (SEQ ID NO:5))+P20(GGGGACCACTTTGTACAAGAAAGCTGGGTATTAAGATGGAGATTTGTG (SEQ ID NO:6)). Theentry clone of CYP716AO21 was recombined into the high copy numberexpression vector pAG423GAL-ccdB (Addgene plasmid 14149) with the GAL1promoter and HIS3 auxotrophic marker, resulting inpAG423[GAL1/CYP716AO21].

Example 9. In Vivo Activity of CYP716AO21 in Yeast Strain TM7

Yeast strain TM7 was generated by super-transforming strain TM3 (seeExample 3) with plasmids pAG423[GAL1/CYP716AO21] andpAG425[GAL1/AtATR1], expressing CYP716AO21 and the A. thaliana CytP450reductase (CPR), AtATR1 (At4g24520), respectively, from the galactoseinducible GAL1 promoter. In parallel, a control strain TM26 harboringonly pAG425[GAL1/AtATR1] in TM3 was also generated. Cell pelletsanalyzed by GC-MS showed the presence of a unique new peak eluting at31.8 minutes in TM7 (FIG. 12, Panel A), but not TM26 (FIG. 12, Panel C).The E1 pattern of this peak corresponded to a hydroxylated derivative ofβ-amyrin, with the alcohol function on either the D or E ring ofβ-amyrin (FIG. 12, Panel D).

Since CYP716AO21 was tentatively annotated as a homolog of the M.truncatula CYP716A12 (GenBank accession number FN995113; (Carelli etal., 2011)), the GC elution time was compared to the E1 pattern of thenew peak in TM7 with a standard of erythrodiol (28-hydroxy-β-amyrin).The strain TM10 was generated by transforming plasmidpAG423[GAL1/CYP716A12] along with pAG425[GAL1/AtATR1] in TM3, andcompared its GC-MS profile with TM7, TM26 and an erythrodiol standard. Apeak corresponding to the elution time and EI of standard erythrodiol(FIG. 12, Panel E) was observed at 32.5 minutes in the GC chromatogramof TM10 (FIG. 12, Panel B) but not TM7 and TM26, indicating thatCYP716AO21 hydroxylates β-amyrin at a position different than CYP716A12.Therefore, the oleanane-type triterpenoid sapogenins found in Bupleurum(Ashour and Wink, 2011) were looked at in order to narrow down thepossible hydroxylation positions of CYP716AO21 on rings D and E ofβ-amyrin to C-16, C-21 and C-29 (FIG. 10), which are positions for whicha CytP450 has not been characterized to date.

Example 10. Effect of CPR:CytP450 Ratio on In Vivo Activity ofCYP716AO21

The endoplasmic reticulum (ER) localized CPRs are flavoproteins,containing both a redox cofactor flavin adenine dinucleotide (FAD) andflavin mononucleotide (FMN), that serve as electron donor proteins forseveral ER oxygenases, including CytP450s. Therefore, optimalinteraction between CPR and CytP450 is essential to allow the reducingequivalents from NADPH to pass from the CPR to the CytP450 (Reed andBackes, 2012). In an attempt to increase the efficiency of hydroxylationof β-amyrin by CYP716AO21, the effect of the ratio of CPR to CytP450 wasdetermined by varying the expression level of AtATR1 while keeping theexpression of CYP716AO21 constant.

The CPR:CytP450 ratios between 1:5 and 1:30 have been reported to beideal for the efficient functioning of yeast and mammalian CytP450s(Reed and Backes, 2012). Therefore, the AtATR1 was expressed from eitheran integrated (pAG305, 1 copy per cell), low-copy number (pAG415, 3-5copies per cell), or high-copy number (pAG425, 10-40 copies per cell)vector, in combination with CYP716AO21 always expressed from thehigh-copy number plasmid (pAG423, 10-40 copies per cell). Thus, twostrains were generated, TM8 and TM9, overexpressingpAG423[GAL1/CYP716AO21] along with pAG305[GAL1/AtATR1] orpAG415[GAL1/AtATR1], respectively, and compared the amount ofhydroxylated β-amyrin produced by these strains with that of TM7. Inaccordance with this assumption, strain TM9 accumulated higher levels ofhydroxylated β-amyrin compared to TM8 and TM7, with the lowestaccumulation in the strain expressing the integrated copy of CPR (FIG.13, Panel A). Therefore, AtATR1 was expressed from the low-copy numbervector pAG415 for further experiments.

Example 11. Secretion of Triterpene Sapogenins from Strains TM9

For the following experiments with strain TM9, condition R2 was employedto avoid the excessive secretion and, hence, loss of β-amyrin, theprecursor for CYP716AO21, from the cells into the medium. Surprisingly,the hydroxylated β-amyrin eluting at 31.8 minutes was only observed inthe GC chromatograms of the spent medium and not cell pellets of TM9upon MβCD treatment (FIG. 13, Panel B), suggesting the completesecretion of the hydroxylated product from the yeast cells into themedium.

The specificity of the type of CD used was also determined on thesequestering of hydroxylated β-amyrin from the cells into the spentmedium of strain TM9. The most abundant variants of CD are α, β and γCD,which have 6, 7 and 8 glucose units, respectively. Therefore, αCD, βCD,γCD, Random MβCD (RMβCD) or MβCD were applied to a final concentrationof 5 mM as in condition R2 and analyzed the spent medium on Day 4, forquantification of the amount of hydroxylated β-amyrin secreted into themedium. Sequestering was only observed with the βCD and its methylatedversions and the highest amount of hydroxylated β-amyrin was detectedupon RMβCD and MβCD treatment, suggesting a strong specificity of themethylated forms of CD over the unmethylated forms, for sequestering oftriterpenoid sapogenins from yeast cells (FIG. 13, Panel C).

Example 12. Transcript Profiling of MeJA-Treated Maesa LanceolataReveals a Novel Plant Cytochrome P450

Maesa lanceolata, a member of the Myrsinaceae family, is a shrub orsmall tree indigenous to Africa. African traditional healers useextracts and/or parts of the plant for the treatment of a wide range ofdiseases including infectious hepatitis, bacillary dysentery, impetigo,ozena, dermatoses and neuropathies. Methanol extracts of M. lanceolataleaves are rich in maesasaponins and have been shown to possessvirucidal, molluscicidal, fungistatic and antimutagenic activities(Sindambiwe et al., 1998). The maesasaponins identified so far arederived from an oleanane skeleton via modifications of the β-amyrinbackbone, resulting in a characteristic C-13,28 hemiacetal or esterbridge and oxidations on C-16, C-21 and C-22 (FIG. 14) (Foubert et al.,2010; Manguro et al., 2011). The hemiacetal or ester bridge between C-13and C-28 is thought to occur through the reaction between a C-13hydroxyl and C-28 aldehyde or carboxyl group, respectively (Vincken etal., 2007). The presence of these diverse oleanane maesasaponinssuggests the presence of a β-amyrin-specific OSC (or β-amyrin synthase)along with specific CytP450s-catalyzing oxygenations at C-16, C-21, C-22and C-28 in M. lanceolata. However, to date, not a single triterpenesaponin biosynthetic gene has been identified from Maesa.

To identify new saponin biosynthetic genes, a transcript profiling wasperformed on methyl jasmonate (MeJA) treated M. lanceolata axenic shootcultures. M. lanceolata axenic shoot cultures were generated andmaintained as described (Faizal et al., 2011). For elicitation, each potof shoot culture was sprayed with 2 ml deionized water containing 0.05%(v/v) TWEEN®-20 in combination with 500 μM MeJA (10 μl of 100 mM stockdissolved in ethanol) or an equivalent amount of ethanol as control. Fortranscript profiling, samples were collected 0, 0.5, 1, 2, 4, 8, 24 and48 hours after elicitor or mock treatments. For each sample, threedifferent plants were pooled.

Using the complete set of 128 BstYI+1/MseI+2 primer combinations, agenome-wide cDNA-AFLP® transcript profiling analysis (Vuylsteke et al.,2007; see also Example 8) was carried out to monitor the expression of atotal of 13,558 transcript tags over time. In total, 733 MeJA-responsivetranscript tags were isolated (hereafter referred to as “ML tags”).Direct sequencing of the reamplified ML tags gave good quality sequencesfor 545 (74.4%) of the fragments. To the remaining 188 tags (25.6%), nounique sequence could be attributed unambiguously, indicating that theymight not represent unique gene tags and, hence, these were notconsidered for further analysis. A BLAST search with the nucleotidesequences of the 545 unique cDNA-AFLP® tags led to the annotation of 312(57.2%) of the ML tags. Average linkage hierarchical clustering analysisof the expression profiles of the ML tags showed that, upon MeJAtreatment, the genes are either transcriptionally activated ortranscriptionally repressed. The activated gene tags can be divided intodifferent subclusters, based on their MeJA response time. In onesubcluster, a gene tag that reached maximum levels of expression 24-48hours post-elicitation and corresponding to squalene epoxidase (SQE) canbe found (FIG. 15). The gene tags ML257 and ML593, corresponding toCytP450s, are tightly co-regulated with this gene (FIG. 15). The genetag ML257 shows homology to the M. truncatula CYP716A12 that was shownto oxidize 3-amyrin in a sequential three-step oxidation on C-28 toyield oleanolic acid through erythrodiol (Carelli et al., 2011;Fukushima et al., 2011), and the gene tag ML593 shows homology to the A.thaliana steroid 22α-hydroxylase gene encoding a CytP450 enzyme thatcatalyzes the oxidation of sterols on the C-22 position (Fujita et al.,2006). The full-length open reading frame ML593 corresponding to therespective gene tag was picked up from a M. lanceolata UncutNanoquantity cDNA library (Pollier et al., 2011), using the primer pairsP27 (CTCTTGCATTCAATCCGAAAC (SEQ ID NO:10))+P28 (AGCAAAGAATGCCTTGGCTA(SEQ ID NO:11)). The FL-ORF of ML593 was PCR amplified for GATEWAY™cloning in pDONR221 using the primer pairs P39(GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGTGGGTAGTGGGATTA (SEQ ID NO:12))+P40(GGGGACCACTTTGTACAAGAAAGCTGGGTATCACTTGTTTTTCTTGGT (SEQ ID NO:13)). Theentry clone ML593 was GATEWAY™ recombined into the high-copy numberexpression vector pAG423GAL-ccdB behind the galactose inducible GAL1promoter and having the HIS3 auxotrophic marker, resulting inpAG423[GAL1/ML593].

Example 13. In Vivo Activity of ML593 in Yeast Strain TM21

To characterize the putative CytP450, ML593 from our transcriptprofiling, strain TM21 was generated from the β-amyrin-producing strainTM3 (Table 4; see Example 3), by supertransforming with the plasmidspAG415[GAL1/AtATR1] and pAG423[GAL1/ML593]. The strains TM21 and TM27(Table 4) were cultured in the presence of MβCD and the spent mediumanalyzed by GC-MS. A new peak eluting at 31.8 minutes corresponding to ahydroxylated β-amyrin in strain TM21 was observed (FIG. 16, Panel A),but not in the control strain TM27 (FIG. 16, Panel C). The EI pattern ofthis peak (FIG. 16, Panel A) corresponded to a hydroxylation on the D orE ring of the oleanane structure and was similar to that observed withstrain TM9 (Table 4) expressing CYP716AO21 (FIG. 16, Panel B). Thestrong similarity between the elution time and EI pattern of thehydroxylated β-amyrin in strain TM21 and TM9 further supports thisassumption. It was also observed that strain TM21 produced eight-foldmore hydroxy β-amyrin than strain TM9, highlighting the betterefficiency of ML593 for hydroxylating β-amyrin as compared toCYP716AO21.

V. Combinatorial Biosynthesis of Triterpenoid Sapogenins in YeastExample 14. Combinatorial Biosynthesis Using CYP716AO21 and CYP716A12

Combinatorial biosynthesis, also known as combinatorial biochemistry,involves the combination of genes from different organisms in aheterologous host to produce bioactive compounds by establishing novelenzyme-substrate combinations in vivo, which, in turn, could lead to thebiosynthesis of novel natural products (Pollier et al., 2011). AlthoughCYP716AO21 is tentatively annotated as a homolog of CYP716A12, the GCelution time and E1 pattern of the β-amyrin hydroxylation product ofCYP716AO21 is different from erythrodiol (FIG. 12). It was reasoned thatif the two enzymes hydroxylate β-amyrin at two different carbonpositions, it should be possible to combine the enzymes in the yeaststrain TM3 and produce a combinatorial compound not produced by eitherof the enzymes alone. Therefore, strain TM30 was generated from TM3 byoverexpressing the plasmids pAG415[GAL1/AtATR1] andpAG423[GAL1/CYP716AO21-T2A-CYP716A12], where CYP716AO21 and CYP716A12are stitched together into a self-processing polyprotein via the 2Aoligopeptide (de Felipe et al., 2006), which is expressed from a singlegalactose inducible GAL1 promoter. The self-processing polyprotein ofCYP716AO21 and CYP716A12 was generated by amplifying the FL-ORF ofCYP716AO21 without a stop codon and having a 3′-overhang of the partialT2A sequence using the primer pair P19

(GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGG AACTTTCTATCACT (SEQ ID NO: 5)) +P23 (ACCGCAUGTTAGCAGACTTCCTCTGCCCTCAGATGGAGATTTGTGGGGAT (SEQ ID NO: 8)).The FL-ORF of CYP716A12 was amplified with a 5′-overhang of the partialT2A sequence using the primers P24

(ATGCGGUGACGTCGAGGAGAATCCTGGCCCAATGGAG CCTAATTTCTATC (SEQ ID NO: 9)) +P22 (GGGGACCACTTTGTACAAGAAAGCTGGGTATTAAGCTTTGTGTGGATAAAGGCG (SEQ ID NO: 7))such that there was an overlap of 7 bp between the two amplifiedsequences. Since the primers P23 and P24 contain an Uracil each, theCYP716AO21 and CYP716A12 were PCR amplified using the Pfu Turbo Cxpolymerase (Stratagene). The purified gel fragments were used forUracil-Specific Excision or USER™ Cloning (New England Biolabs) togenerate two fragments with complementary sticky ends that were ligatedin vitro using the T4 DNA ligase (Invitrogen). The ligated DNA productwas once again gel purified and used as template for amplification withthe primers P19+P22. This amplicon was GATEWAY™ recombined intopDONR221, sequence verified and further recombined into pAG423GAL-ccdBto generate pAG423[GAL1/CYP716AO21-T2A-CYP716A 12].

The spent medium of strain TM30 cultured in the presence of MβCD wasanalyzed by GC-MS and compared to the GC chromatograms of spent mediumfrom strains TM9, and TM17 overexpressing pAG423[GAL1/CYP716A12] andpAG415[GAL1/AtATR1] in TM3. A unique peak was observed at 40.5 minutesin strain TM30 (FIG. 17, Panel A) but not TM9 (FIG. 17, Panel B) andTM17 (FIG. 17, Panel C), strongly supporting the fact that CYP716AO21and CYP716A12 catalyze hydroxylations of two different carbons onβ-amyrin. Additionally, the EI pattern of this peak suggested thepresence of carboxyl and alcohol functions on β-amyrin, indicating theC-28 carboxylation by CYP716A12 and the C-16, C-21 or C-29 hydroxylationby CYP716AO21. Considering the close proximity of C-16 and C-28 on theβ-amyrin molecule (FIG. 10) and the tentative annotation of CYP716AO21as a homolog of CYP716A12, it was reasoned that CYP716AO21 mighthydroxylate C-16 of β-amyrin. Therefore, the GC chromatogram and E1pattern of an echinocystic acid standard (FIG. 17, Panel E) werecompared with that of the new peak at 40.5 minutes in TM30 (FIG. 17,Panel D). However, the GC elution time and fragmentation of the new peakat 40.5 minutes did not match that of echinocystic acid(3β,16α-dihydroxyolean-28-oic acid), ruling out the possibility of aC-16 α-hydroxylation by CYP716AO21. Due to the absence of authentic3β,16β-dihydroxyolean-28-oic acid, 3β,21β-dihydroxyolean-28-oic acid and3β,29α-dihydroxyolean-28-oic acid standards, the identity of thecombinatorial compound was unable to be confirmed.

In conclusion, using a combinatorial approach in S. cerevisiae, it wasrevealed that CYP716AO21 is not a C-28 oxygenase but a novel cytochromeP450 that encodes a β-amyrin 16β/21β/29α-hydroxylase involved in thesynthesis of saikosaponins in Bupleurum.

Example 15. Combinatorial Biosynthesis Using ML593 and CYP716A12

First, to confirm the identity of ML593 as a functional homolog ofCYP716AO21, strain TM31 was generated, similar to strain TM30 (Table 4)(see Example 14). Strain TM31 was created by supertransforming TM3 withthe plasmids pAG415[GAL1/AtATR1] and pAG423[GAL1/ML593-T2A-CYP716A12],where ML593 and CYP716A12 form a self-processing polyprotein stitchedtogether by the 2A oligopeptide (de Felipe et al., 2006) and isexpressed from a single GAL1 promoter. The strains TM31, TM17 and TM21(Table 4) were cultured in the presence of MβCD and the spent medium wasanalyzed using GC-MS. Similar to strain TM30 (FIG. 18, Panel D), a newpeak was observed eluting at 40.5 minutes in strain TM31 (FIG. 18, PanelA), but not the strains TM17 (FIG. 18, Panel B) and TM21 (FIG. 17, PanelC) expressing only the CytP450 CYP716A12 and ML593, respectively. Fromthe E1 pattern of this peak, its identity was confirmed as being thesame as in strain TM30, which, together with the lack of homology withechinocystic acid, suggests a β-hydroxylation on C-21, the onlyremaining position commonly hydroxylated between CYP716AO21 and ML593.Although the CYP716AO21 and ML593 belong to different CytP450subfamilies (CYP716A and CYP90B, respectively), in the yeast straindisclosed herein demonstrates the same catalytic activity.

Example 16. Combinatorial Biosynthesis Using ML593 and CYP88D6

To generate a combinatorial scaffold using β-amyrin, ML593 (see Examples12 and 13) and CYP88D6 were expressed in strain TM3. The CytP450 CYP88D6(GenBank accession number AB433179; (Seki et al., 2008)) catalyzes atwo-step oxidation of β-amyrin to 11-oxo-β-amyrin through an11-hydroxy-β-amyrin intermediate. First, the activity of CYP88D6 wasconfirmed in the β-amyrin-producing strain by generating strain TM18(Table 4) and culturing it in the presence of MβCD along with thecontrol strain TM27. Therefore, the full-length open reading frame(FL-ORF) of CYP88D6 was PCR amplified using the primers P45(GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGAAGTACATTGGGTTTG (SEQ ID NO:16))+P46(GGGGACCACTTTGTACAAGAAAGCTGGGTACTAAGCACATG AAACCTTTA (SEQ ID NO:17)) andcloned into pDONR221. The CytP450 CYP88D6 was then GATEWAY™ recombinedinto the high-copy number expression vector pAG423GAL-ccdB (Addgeneplasmid 14149). GC chromatograms of the spent medium of strain TM18showed the presence of four unique peaks (FIG. 19, Panel C) that wereabsent in the control strain TM27 (FIG. 19, Panel D). Two of these peakseluting at 25.4 minutes and 37.5 minutes corresponded to 11-hydroxyβ-amyrin and 11-oxo β-amyrin (Seki et al., 2008), respectively. The tworemaining peaks eluting at 24.4 minutes and 26.6 minutes could not beassigned an identity despite their clear EI pattern. The highest massobserved in the mass spectra extracted from these peaks (FIG. 19, PanelE) was lower than that of trimethylsilylated β-amyrin (M+=498),indicating their possible non-triterpenoid origin. These additionalpeaks observed in our yeast strain were not reported when the CYP88D6was expressed in a wild-type yeast strain expressing a β-amyrin synthasefrom Lotus japonicus (Seki et al., 2008).

Further, the strain TM32 was generated by supertransforming strain TM3with the plasmids pAG415[GAL1/AtATR1] andpAG423[GAL1/ML593-T2A-CYP88D6], where ML593 and CYP88D6 are stitchedtogether with the 2A oligopeptide, resulting in the generation of aself-processing polypeptide (de Felipe et al., 2006). Theself-processing polyprotein of ML593 and CYP88D6 was generated byamplifying the FL-ORF of ML593 without a stop codon and having a3′-overhang of the partial T2A sequence using the primer pair P39

(GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGTG GGTAGTGGGATTA (SEQ ID NO: 12)) +P47 (ACCGCAUGTTAGCAGACTTCCTCTGCCCTCCTTGTTTTTCTTGGTGACCT (SEQ ID NO: 18)).The FL-ORF of CYP88D6 was amplified with a 5′-overhang of the partialT2A sequence using the primers P48

(ATGCGGUGACGTCGAGGAGAATCCTGGCCCAATGGA AGTACATTGGGTTT (SEQ ID NO: 19)) +P46 (GGGGACCACTTTGTACAAGAAAGCTGGGTACTAAGCACATGAAACCTTTA (SEQ ID NO: 17)),such that there was an overlap of 7 bp between the two amplifiedsequences. Since the primers P48 and P47 contain a Uracil each, theML593 and CYP88D6 were PCR amplified using the Pfu Turbo Cx polymerase(Stratagene). The purified gel fragments were used for USER™ Cloning(New England Biolabs) to generate two fragments with complementarysticky ends, which were ligated in vitro using the T4 DNA ligase(Invitrogen). The ligated DNA product was used as template foramplification with the primers P39+P46. This amplicon was GATEWAY™recombined into pDONR221, sequence verified and further recombined intopAG423GAL-ccdB to generate pAG423[GAL1/ML593-T2A-CYP88D6]. The spentmedium of strain TM32 (FIG. 19, Panel A) cultured in the presence ofMβCD was compared with that of strains TM21 (FIG. 1, Panel B) TM18 (FIG.19, Panel C), and TM27 (FIG. 19, Panel D). Strain TM32 showed thepresence of two unique peaks eluting at 32.1 minutes and 44.7 minutesthat could correspond to 11α,21β(?)-dihydroxy β-amyrin and11-oxo-21β(?)-hydroxy β-amyrin based on their EI pattern, respectively.

Example 17. Combinatorial Biosynthesis Using ML593 and CaDDS

In an attempt to generate a dammarenediol-producing yeast strain, theplasmid pESC-URA[GAL10/tHMG1; GAL1/CaDDS] was expressed, harboring adammarenediol synthase gene (CaDDS) from Centella asiatica (GenBankaccession number AY520818; (Kim et al., 2009)), in the sterol-modifiedyeast strain TM1, to generate strain TM33. The CaDDS was amplified withXhoI and NheI containing primers P43 (GGGGACAAGTTTGTACAAAAAAGCAGGCTTActcgagATGTGGAAGCTGAAGATAGCA (SEQ ID NO:14))+P44(GGGGACCACTTTGTACAAGAAAGCTGGGTTgctagcTCAATTGGAGAGCCACAAGCG (SEQ IDNO:15)) to generate pESC-URA[GAL10/tHMG1; GAL1/CaDDS].

Strain TM33 and the control strain TM5 was cultured in medium containingMβCD and then analyzed the GC chromatograms obtained from the spentmedium of both the strains. Unexpectedly, three new peaks were found instrain TM33 eluting at 27.2 minutes, 28.6 minutes, and 33.5 minutes(FIG. 20, Panel A) that were absent in the control strain TM5 (FIG. 20,Panel B). The identity of the peak eluting at 27.2 minutes was confirmedas β-amyrin (FIG. 20, Panel C), based on its elution time and its EIpattern. The peak at 28.6 minutes had a similar EI pattern as β-amyrinand was confirmed as α-amyrin by comparing to a standard (FIG. 20, PanelD). The E1 pattern of the peak at 33.5 minutes could be interpreted todammarenediol-II (Spencer, 1981), but was not confirmed due to the lackof an authentic standard. Although, the CaDDS was initially reported asa putative bAS (Kim et al., 2005) and later characterized as adammarenediol synthase (Kim et al., 2009), in the yeast strain disclosedherein, the gene was capable of cyclizing 2,3-oxidosqualene to bothβ-amyrin and dammarenediol in addition to a third product, α-amyrin. Inthe yeast strain disclosed herein, the relative amounts of α-amyrin,β-amyrin and dammarenediol-II were in the ratio of 8.8:1.1:0.1,highlighting the very low dammarene synthase activity of CaDDS asopposed to its current characterization.

Strain TM37 was generated from strain TM33 by supertransforming with theplasmids pAG415[GAL1/AtATR1] and pAG423[GAL1/ML593] (see Table 4). TheML593 was characterized as a putative C-21 hydroxylase of β-amyrin (seeExamples 12 and 13) and the substrate specificity of this CytP450 wasdetermined by expressing it together with the multifunctional cyclaseCaDDS. Strain TM37 and the control strain TM38 were cultured in thepresence of MβCD and compared GC chromatograms for the presence ofunique peaks. Two peaks were identified eluting at 31.8 minutes and 33.2minutes in the spent medium of TM37 (FIG. 21, Panel A), but not thecontrol strain TM38 (FIG. 21, Panel B). The E1 pattern of both thesepeaks were identical (FIG. 21, Panel A), and the peak at 31.8 minutescorresponded to (most likely) 21-hydroxy β-amyrin (see Examples 12 and13). Therefore, the second peak at 33.2 minutes could correspond to21-hydroxy α-amyrin, but was not confirmed due to the absence of anauthentic standard.

TABLE 4 List of yeast strains generated and used in this study. NameConstruct S288c BY4742 MATa; his3Δ1; leu2Δ0; ura3Δ0; lys2Δ0 TM1 S288cBY4742; P_(erg7)::P_(MET3)-ERG7 TM3 TM1; pESC-URA[GAL10/tHMG1;GAL1/GgbAS](36.2 mg/L (β-amyrin) TM5 TM1; pESC-URA[GAL10/tHMG1] TM7 TM3;pAG423[GAL1/CYP716AO21], pAG425[GAL1/AtATR1] TM8 TM3;pAG423[GAL1/CYP716AO21], pAG305[GAL1/AtATR1] TM9 TM3;pAG423[GAL1/CYP716AO21], pAG415[GAL1/AtATR1] TM10 TM3;pAG423[GAL1/CYP716A12], pAG425[GAL1/AtATR1] TM17 TM3;pAG423[GAL1/CYP716A12], pAG415[GAL1/AtATRI] TM21 TM3;pAG423[GAL1/ML593], pAG415[GAL1/AtATR1] TM26 TM3; pAG423,pAG425[GAL1/AtATR1] TM30 TM3; pAG423[GAL1/CYP716AO21-T2A-CYP716Al2],pAG415[GAL1/AtATR1] TM27 TM3; pAG423, pAG415[GAL1/AtATR1] TM31 TM3;pAG423[GAL1/ML593-T2A-CYP716A12], pAG415[GAL1/AtATR1] TM18 TM3;pAG423[GAL1/CYP88D6], pAG415[GAL1/AtATR1] TM32 TM3;pAG423[GAL1/ML593-T2A-CYP88D6], pAG415[GAL1/AtATR1] TM33 TM1;pESC-URA[GAL10/tHMG1; GAL1/CaDDS] TM37 TM33; pAG423[GAL1/ML593],pAG415[GAL1/AtATR1] TM38 TM33; pAG423, pAG415[GAL1/AtATR1]

TABLE 5 Sequences of primers. Sequence SEQ Primer (5′ to 3′) ID NO P19GGGGACAAGTTTGTACAAAAAAGCAGG 5 CTTAATGGAACTTTCTATCACT P20GGGGACCACTTTGTACAAGAAAGCTGG 6 GTATTAAGATGGAGATTTGTG P22GGGGACCACTTTGTACAAGAAAGCTGG 7 GTATTAAGCTTTGTGTGGATAAAGGCG P23ACCGCAUGTTAGCAGACTTCCTCTGCC 8 CTCAGATGGAGATTTGTGGGGAT P24ATGCGGUGACGTCGAGGAGAATCCTGG 9 CCCAATGGAGCCTAATTTCTATC P27CTCTTGCATTCAATCCGAAAC 10 P28 AGCAAAGAATGCCTTGGCTA 11 P39GGGGACAAGTTTGTACAAAAAAGCAGG 12 CTTAATGTGGGTAGTGGGATTA P40GGGGACCACTTTGTACAAGAAAGCTGG 13 GTATCACTTGTTTTTCTTGGT P43GGGGACAAGTTTGTACAAAAAAGCAGG 14 CTTActcgagATGTGGAAGCTGAAGAT AGCA P44GGGGACCACTTTGTACAAGAAAGCTGG 15 GTTgctagcTCAATTGGAGAGCCACAA GCG P45GGGGACAAGTTTGTACAAAAAAGCAGG 16 CTTAATGGAAGTACATTGGGTTTG P46GGGGACCACTTTGTACAAGAAAGCTGG 17 GTACTAAGCACATGAAACCTTTA P47ACCGCAUGTTAGCAGACTTCCTCTGCC 18 CTCCTTGTTTTTCTTGGTGACCT P48ATGCGGUGACGTCGAGGAGAATCCTGG 19 CCCAATGGAAGTACATTGGGTTT

The sequences in lower case represent the restriction recognition siteused for restriction enzyme-mediated cloning. The underlined sequencecorresponds to T2A partial sequences.

TABLE 6 List of sequences SEQ ID NO Nucleotide sequence CYP716AO21 1ATGGAACTTTCTATCACTCTGATGCTTA TTTTCTCAACAACCATCTTCTTTATATTTCGTAATGTGTACAACCATCTCATCTCT AAACACAAAAACTATCCCCCTGGAAGTATGGGCTTGCCTTACATTGGCGAAACACT TAGTTTCGCGAGATACATCACCAAAGGAGTCCCTGAAAAATTCGTAATAGAAAGAC AAAAGAAATATTCAACAACAATATTTAAGACCTCCTTGTTCGGAGAAAACATGGTG GTGTTGGGCAGTGCAGAGGGCAACAAATTTATTTTTGGAAGCGAGGAGAAGTATTT ACGAGTGTGGTTTCCAAGTTCTGTGGACAAAGTGTTCAAAAAATCTCATAAGAGAA CGTCGCAGGAAGAAGCTATTAGGTTGCGCAAAAACATGGTGCCATTTCTCAAAGCA GATTTGTTGAGAAGTTATGTACCAATAATGGACACATTTATGAAACAACATGTGAA CTCGCATTGGAATTGCGAGACCTTGAAGGCTTGTCCTGTGATCAAGGATTTTACGT TTACTTTAGCTTGTAAACTTTTTTTTAGTGTAGACAATCCTTTGGAGCTAGAGAAG TTAATCAAGCTATTTGTGAATATAGTGAATGGCCTCCTTACGGTCCCTATTGATCT CCCGGGGACAAAATTTAGAGGAGTTATAAAGAGTGTCAAGACTATTCGCCATGCGC TTAAAGTGTTGATCAGGCAACGAAAGGTGGATATTAGAGAGAAAAGAGCCACACCT ACGCAAGATATATTGTCGATAATGCTGGCACAGGCTGAGGACGAGAACTATGAAAT GAATGATGAAGATGTGGCCAATGACTTTCTTGCAGTTTTGCTTGCTAGTTATGATT CTGCCAATACTACACTCACCATGATTATGAAATATCTTGCTGAATATCCCGAAATG TATGATCGAGTTTTCAGAGAACAAATGGAGGTGGCAAAGACGAAAGGAAAAGATGA ATTACTCAACTTGGACGACTTGCAAAAGATGAATTATACTTGGAATGTAGCTTGTG AAGTACTGAGAATTGCAACACCAACGTTCGGAGCATTCAGAGAGGTTATTGCAGAT TGTACATACGAAGGGTACACCATACCAAAAGGCTGGAAGCTATATTATGCCCCGCG TTTTACCCATGGAAGTGCAAAATACTTTCAAGATCCAGAGAAATTTGATCCATCGC GATTTGAAGGTGATGGTGCGCCTCCTTATACATTCGTTCCATTCGGAGGAGGGCTC CGGATGTGCCCTGGATACAAGTATGCAAAGATTATAGTACTAGTGTTCATGCACAA TATAGTTACAAAGTTCAAATGGGAGAAAGTTAACCCTAATGAGAAAATGACAGTAG GAATCGTATCAGCGCCAAGTCAAGGACTTCCACTGCGTCTCCATCCCCACAAATCT CCATCTTAA ML593 2ATGTGGGTAGTGGGATTAATTGGTGTGG CTGTGGTAACAATATTGATAACTCAGTATGTATACAAATGGAGAAATCCAAAGACT GTGGGTGTTCTGCCACCTGGTTCAATGGGTCTGCCTTTGATCGGGGAGACTCTTCA ACTTCTCAGCCGTAATCCATCCTTGGATCTTCATCCTTTCATCAAGAGCAGAATCC AAAGATATGGGCAGATATTCGCGACCAATATCGTAGGTCGACCCATAATAGTAACC GCTGATCCGCAGCTCAATAATTACCTTTTCCAACAAGAAGGAAGAGCAGTAGAACT GTGGTACTTGGACAGCTTTCAAAAGCTATTTAACTTAGAAGGTGCAAACAGGCCGA ACGCAGTTGGTCACATTCACAAGTACGTTAGAAGTGTATACTTGAGTCTCTTTGGC GTCGAGAGCCTTAAAACAAAGTTGCTTGCCGATATTGAGAAAACAGTCCGCAAAAA TCTTATTGGTGGGACAACCAAAGGCACCTTTGATGCAAAACATGCTTCTGCCAATA TGGTTGCTGTTTTTGCTGCAAAATACTTGTTCGGACATGATTACGAGAAATCGAAA GAAGATGTAGGCAGCATAATCGACAACTTCGTACAAGGTCTTCTCGCATTCCCATT GAATGTTCCCGGTACAAAGTTCCACAAATGTATGAAGGACAAGAAAAGGCTGGAAT CAATGATCACTAACAAGCTAAAGGAGAGAATAGCTGATCCGAACAGCGGACAAGGG GATTTCCTTGATCAAGCAGTGAAAGACTTGAATAGCGAATTCTTCATAACAGAGAC TTTTATCGTTTCGGTGACGATGGGAGCTTTATTTGCGACGGTTGAATCGGTTTCGA CAGCAATTGGACTAGCTTTCAAGTTTTTTGCAGAGCACCCCTGGGTTTTGGATGAC CTCAAGGCTGAGCATGAGGCTGTCCTTAGCAAAAGAGAGGATAGAAATTCACCTCT CACGTGGGACGAATATAGATCGATGACACACACGATGCACTTTATCAATGAAGTCG TCCGTTTGGGAAATGTTTTTCCTGGAATTTTGAGGAAAGCACTGAAAGATATTCCA TATAATGGTTATACAATTCCGTCCGGTTGGACCATTATGATTGTGACCTCTACCCT TGCGATGAACCCTGAGATATTCAAGGATCCTCTTGCATTCAATCCGAAACGTTGGC GGGATATTGATCCCGAAACTCAAACTAAAAACTTTATGCCTTTCGGTGGTGGGACG AGACAATGCGCAGGTGCAGAGCTAGCCAAGGCATTCTTTGCTACCTTCCTCCATGT TTTAATCAGCGAATATAGCTGGAAGAAAGTGAAGGGAGGAAGCGTTGCTCGGACAC CTATGTTAAGTTTTGAAGATGGCATATTTATTGAGGTCACCAAGAAAAACAAGTGA Amino acid sequence CYP716AO21 3MELSITLMLIFSTTIFFIFRNVYNHLIS KHKNYPPGSMGLPYIGETLSFARYITKGVPEKFVIERQKKYSTTIFKTSLFGENMV VLGSAEGNKFIFGSEEKYLRVWFPSSVDKVFKKSHKRTSQEEAIRLRKNMVPFLKA DLLRSYVPIMDTFMKQHVNSHWNCETLKACPVIKDFTFTLACKLFFSVDNPLELEK LIKLFVNIVNGLLTVPIDLPGTKFRGVIKSVKTIRHALKVLIRQRKVDIREKRATP TQDILSIMLAQAEDENYEMNDEDVANDFLAVLLASYDSANTTLTMIMKYLAEYPEM YDRVFREQMEVAKTKGKDELLNLDDLQKMNYTWNVACEVLRIATPTFGAFREVIAD CTYEGYTIPKGWKLYYAPRFTHGSAKYFQDPEKFDPSRFEGDGAPPYTFVPFGGGL RMCPGYKYAKIIVLVFMHNIVTKFKWEKVNPNEKMTVGIVSAPSQGLPLRLHPHKS PS ML593 4 MWVVGLIGVAVVTILITQYVYKWRNPKTVGVLPPGSMGLPLIGETLQLLSRNPSLD LIAPFIKSRIQRYGQIFATNIVGRPIIVTADPQLNNYLFQQEGRAVELWYLDSFQK LFNLEGANRPNAVGHIHKYVRSVYLSLFGVESLKTKLLADIEKTVRKNLIGGTTKG TFDAKHASANMVAVFAAKYLFGHDYEKSKEDVGSIIDNFVQGLLAFPLNVPGTKFH KCMKDKKRLESMITNKLKERIADPNSGQGDFLDQAVKDLNSEFFITETFIVSVTMG ALFATVESVSTAIGLAFKFFAEHPWVLDDLKAEHEAVLSKREDRNSPLTWDEYRSM THTMHFINEVVRLGNVFPGILRKALKDIPYNGYTIPSGWTIMIVTSTLAMNPEIFK DPLAFNPKRWRDIDPETQTKNFMPFGGGTRQCAGAELAKAFFATFLHVLISEYSWK KVKGGSVARTPMLSFEDGIFIEVTKKNK

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The invention claimed is:
 1. A method for the intracellular productionand extracellular secretion of triterpenoid sapogenins in a geneticallyengineered yeast culture, the method comprising: incubating geneticallyengineered yeast cells that express an exogenous regulatory orbiosynthetic enzyme of the sapogenin biosynthesis pathway in a culturemedium comprising randomly methylated cyclodextrins, hydroxypropylatedcyclodextrins, and/or beta-cyclodextrins at a concentration such thatthe cyclodextrins increase the amount of the produced triterpenoidsapogenin secreted from the eukaryotic genetically engineered yeastcells into the culture medium in comparison to a culture medium lackingsaid cyclodextrins where the biosynthetic enzyme is an oxidosqualanecyclase and/or a cytochrome P450.
 2. The method according to claim 1,wherein the yeast cells are Saccharomyces cells, Schizosaccharomycescells, Pichia cells, Yarrowia cells, Hansenula cells, Kluyveromycescells, or Candida cells.
 3. The method according to claim 1, wherein thegenetically engineered yeast cells are deficient in expression and/oractivity of an enzyme involved in endogenous sterol synthesis.
 4. Themethod according to claim 1, wherein the culture medium comprisescyclodextrins selected from the group consisting of randomly methylatedcyclodextrins and hydroxypropylated cyclodextrins.
 5. The methodaccording to claim 1, wherein the culture medium comprisesβ-cyclodextrin.
 6. The method according to claim 1, wherein thecyclodextrin concentration in the culture medium is less than 25 mM. 7.The method according to claim 1, wherein cyclodextrins are added to theculture medium at different consecutive time points.
 8. The methodaccording to claim 1, further comprising: extracting sapogenins from theculture medium.
 9. The method according to claim 1, wherein the yeastcells are genetically engineered to overexpress an oxidosqualene cyclaseand/or a cytochrome P450.
 10. The method according to claim 6, whereinthe cyclodextrin concentration is less than 10 mM.
 11. The methodaccording to claim 10, wherein the cyclodextrin concentration is between2 mM and 7 mM.